Berk2010-Tahoura: Difference between revisions

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=[[User:Tahoura Samad|Tahoura Samad]] 12:55, 25 June 2010 (EDT)=
*[[Robot]]
==PDD Subgroup Work==
*[[Image:20101118 783.JPG|100px]]
*got 6 with 2348 and 6 with 2349 out of shaker at around 10 am. Check with Jin when we can look at them under the microscope.  
*[[June]]
*miniprepped pBca1601-Bjh2291.
 
*Started transformations.  
 
=[[User:Tahoura Samad|Tahoura Samad]] 15:18, October 15  2010 (EDT)=
-went down to Koshland to do confocal with Scott from the king lab. Could only load 30 ul of sample at time. Looked at NLS and 51 after induction fifteen minutes after feeding.
-saw one possible hit, but set up wasn't optimal
-choanos were suspended in low melt temp agarose.
-probably better to use scope in Stanley.
-lysis looks like it occured in pmll KC. miniprepped the plasmid, but probably will not use because it took so long. Also streaked out control, and will retest for lysis
-for the future: JTK030 and 159 have different copy numbers. Probably why timing is different for 159 with 2801 and NLS which is 030.
-New lifeact part 171 in 1600 still did not grow up
 
=[[User:Tahoura Samad|Tahoura Samad]] 15:18, October 14 2010 (EDT)=
-started fresh choano culture by adding Kan to choano media culture.
-picked colonies of AKS NLS with 51 and 52 as well as 2801 with 51 and 52 to look at under Koshland confocal.
-checked on colonies of iGEM171 in 1600 (p15 spec). Did not grow up. Talk to Tim.
-tested lysis of PCR of 51 digested and ligated in pmll KC. Did not appear to have lysed after 6 hours. kept in shaker
 
=[[User:Tahoura Samad|Tahoura Samad]] 15:18, October 13  2010 (EDT)=
-set up test with NLS in 030 for 51 and 52 for confocal in upstairs stanley.
-52 still behaving oddly, with little hits/fluoresence
-got an excellent image of 51 at timepoint -15. showed fluoresence clearly filling a choano shaped object. YIPEEEEEE!!!
-controls for new 11 cells looked clean. Transformations grew up alright.
-controls for Daniela's 1600 payload cells looked clean.
 
=[[User:Tahoura Samad|Tahoura Samad]] 15:18, October 12 2010 (EDT)=
-made competent cells of 11 and transformed them with 51 and 52 ran controls on AC, S, K.
 
=[[User:Tahoura Samad|Tahoura Samad]] 15:18, October 11 2010=
-restransformed 51 . 52 into 9.
 
=[[User:Tahoura Samad|Tahoura Samad]] 15:18, 6 October 2010 (EDT)=
-checked lysis Tecan with Daniela for iGEM171 with 51 and 52 in 1256. Appears to have lysed, just slowly. Tim is E/Bing it into p15a vector
 
=[[User:Tahoura Samad|Tahoura Samad]] 15:18, 5 October 2010 (EDT)=
-fed choanos
=[[User:Tahoura Samad|Tahoura Samad]] 15:18,  4 October 2010 (EDT)=
-did choano assays with 1934 NLS in JTK030 at induction time points +30, +15, 0, -15.  
-Had dones more hits in 51 for 0 and -15 time points. could barely move in field of view without seeing a hit!
-52 did not have any hits at all.
 
 
 


=[[User:Tahoura Samad|Tahoura Samad]] 14:15, 24 June 2010 (EDT)=
[[Image:20100915 204.JPG]]=
==PDD Subgroup Work==
=[[User:Tahoura Samad|Tahoura Samad]] 15:18, 24 September 2010 (EDT)=
*picked AK transformations of Bjh2291.
*Started Eco/Bam of stuff into CK.  
*Transformations failed again. Tests for competent cell life looked good. 1, 2, 3 with Bjh2331, 2333, 2348, 2349 failed because i plated them on Cam when they are not Cam resistant.  :( 7 probably failed because it just sucks. Ask Jin though. Jin says not to worry about 7 because we have many other possible combinations. Jin also says he would be able to look at the final combinations on Monday. Keep at room temperature after incubation?
*Mass production of choanos.
*in control plates to see if competent cell life was good, there were multiple phenotypes, red and small and big and white. Did colony PCR on two samples from 1 and two samples from 7 with ca998 and g00101 to see if these were actually cells, or were contamination. Colony PCR showed 3 strong bands at 3 kb, which is what we expected.
*looked at choanoes ater shaking at 37 C for almost a full day. All the choanos were dead.
poured plates with TIm.


=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 23 2010 (EDT)=
*Ran assay with 51 and 52 in '8', which is Bjh1255 in JTK030, and '9' which is Bjh1934 in JTK030. Also tried to run Conor's 52 with iGEM159.
*Didn't see invitro lysis for anything in 8, or with Conor's new lifeact construct.
*Will transform over weekend so Tim can rerun assay on Wednesday.
==To Do==
==To Do==
*miniprep 2291
*Start mass production of choanos to run Josh's plasmids and Tera's plasmids.
*transform 2291, 2331,2333,2348,2349 into 1,2,3 again. Plate on K,A, top plate magnesium spec. transform 2291 into 5,6,7,(AKC, top plate spec and magnesium.  
*Test lysis of Amy's CK stuff
*Ask
 
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 22 2010 (EDT)=
*Ran lysis of Amy's Stuff (51 and 52) which did not lyse after 2 hours
*Tried to do lysis at room temperature. Induce at 11 am, and did not see lysis at 6 pm. Looks like we may have had lysis by the next day at around 10 am.
*picked stuff for choano assays again tomorrow. 4 colonies each because of poor growth seen on Monday.
 
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 21 2010 (EDT)=
*Choano care
*ReKan'd a couple plates to get rid of biofilms.
 
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 20  2010 (EDT)=
*Daniela accidently induced same sample twice, so scrapped '8' and '9' assay.  
*Ran choano assay on just 1968 in '11' with plain '11' as control. Didn't really see any of the same successful choano phenotype after 3 hours after feed at 90,60 and 30 min induction points. In addition, adding more bacteria (5ul vs 1ul) doesn't really appear to help efficiency. Just see a lot of stuff lysing outside of choanos. May not be limiting factor.


=[[User:Tahoura Samad|Tahoura Samad]] 19:20, 23 June 2010 (EDT)=
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 19 2010 (EDT)=
==PDD Subgroup Work==
*picked stuff for choano assay on Monday which includes 51 and 52 in 8 and 9, as well as Bjh1968, just self-lysis.
*checked on 18 transformations. All failed except 6 with 2348, 6 with 2349 which had poor growth, 1 colony and two colonies respectively. Result was unexpected since growth was fairly good for the 6 previous plates. Possible reasons: plates were badNot a problem with miniprep, because it worked for first 6 transformations.
 
*redid all 18 transformations.  
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 18 2010 (EDT)=
*Also, transformed and plated E/B transfer of 2291.
*played with new iGEM friends with Christoph.  
*started choano work. Conor filtered 12 mL of choanoes. We got a 12 well plate. We added 360 μL of I M Mgs04 to 12 mL of choanoes. We put1 mL of choanoes into each of 6 wells, added 60 μL of NH4Cl to the remaining 6 mL and put these into the other 6 wells. We then added 10 μL of the TB culture into each of the wells. The plate needs to sit in the incubator for 2 to 3 hours.
*Out of five, only one, the Tetrahymena, appeared to have eaten the bacteria within the 4 hours we looked at them. At the final hour, samples were sluggish and swimming in erratic circles=not healthy.
*Concerned about size being too big to see lysis even if it was occuring.
 
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 17 2010 (EDT)=
*slept through self-lysis assay induction points. Will run on Sat or Monday.
 
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 14 2010 (EDT)=
*ran same choano assay again. SUCESSS!!! Details in spreasheet. :D
Induction timepoints: 


==To Do==
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 10 2010 (EDT)=  
*pick colonies up if they grow. Grow up in TB and magnesium at the end of the day.
*Look at choanos again after about 24 hours. FP signal is reallly low, takes about 15000 ms to really pick up anything in lysed samples. Not a lot of bacteria anywhere, especially in lysed samples.  
*test cam/spec plates I plated at some point to see if they are any good.
*Had Christoph transform again.
*ask Terry how to use the microscope downstairs in Stanley
*BART tickets.
*pick 2291 and grow up
*miniprep 2291 on Friday. Transform in 6 differnet cell lines on Monday.  
*pick and grow up on Tuesday


=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 9 2010 (EDT)=
*Ran choano assay. Induction starting at 10:15, with time points at 90,75,60,45,30,15. Fed at 12:00, So block was in shaker 15 minutes longer than expected. Started looking at approximately 3 pm.
*GFP=300ms
Phase=162 ms
*SUCCESS!!!!!! Saw green choanos in 51 at 90, 75 time points. Other samples were photobleached. Will save samples to look at again tomorrow and see if FPs last.


=[[User:Tahoura Samad|Tahoura Samad]] 12:57, 22 June 2010 (EDT)=
==PDD Subgroup Work==
*checked  controls for competent BAC with Bjh2343. Looked good, with good growth on Cam/Spec, and no growth on Kan, Amp.
[[Image:IMG_4961.JPG|100px]]positive control for Cam/Spec<br>
[[Image:IMG_4960.JPG|100px]]negative control for Amp<br>
[[Image:IMG_4959.JPG|100px]]negative control for Kan<br>
*started transformations of PDDs into competent cells. Did Bjh2331, Bjh2333, Bjh2348, Bjh2349 into BAC with pBca1256-Bjh2343, pBca1256-Bjh2343, pBca1256-Bjh2344, pBca1256-Bjh1934, and redid BAC with pBca1256-Bjh2344 with Bjh2348 and 2349. (18)
*picked colonies for assay. Need to grow for 16 hours, so grow late in the day. Grow in TB with four antibiotics. started at 5:24 pm. 16 hrs is 9:24 am.
*throughout Tim's ecoli, because transducing them messed up the ligation part of the process, so they were useless.
*started Eco/Bam of Bjh2291 up to ligation. Transform tomorrow.
2331:2343 w BAC:7
2333:2343 w BAC:7
2348:2343 w BAC:7
2349:2343 w BAC:7


2331:2343
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 8 2010 (EDT)=
2333:2343
*picked colonies to assay tomorrow. Grew up in TB. Will aspirate off ASW, and then resuspend in choano media.
2348:2343
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 7 2010 (EDT)=
2349:2343
*Set up TECAN with LB, LB culture resuspended in ASW, Choano, 50:50, 25:75, 75:25 LB:ASW, and same for TB, TB resuspended.... S
*Graphs show clear lysis for everything except for ASW and 50:50 for both 51 and 52.
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 6 2010 (EDT)=
*Christoph ran lysis assays over weekend in LB/ASW and choano media. Said he saw lysis in all of them. Will run Tecan tomorrow to confirm.
*Did microscopy assay of 51 and 52 induced 90, 75, 60,45,30,15 mins ahead of time after 24 hours. Did not see green phenotype anywhere, or really all that much green. Not a success, but not surprising considering that we don't really see lysis in ASW.


==To Do==
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 3 2010 (EDT)=  
*microscope assays
*Microscopy comparing Tb vs LB 1, 2, 5, 10 ul.
*transform and plate 2291 in MC1061 and MG1655.  
*Ran PCR of Pcons with Lifeact FP.
*transform 2291 into all 6 different strains.
*Gave payload project to conor.
*depending on tomorrow, pour more 4 antibiotic plates.  
*Ran quick macroscale assay of lysis in ASW. Not apparent after four hours.  


=[[User:Tahoura Samad|Tahoura Samad]] 15:18, 21 June 2010 (EDT)=
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 2 2010 (EDT)=  
==PDD Sugroup Work==
*Had Christoph induce cells, started assaying at 3:00. Fatal error. Not enough bacteria for cells to eat. Will do a concentration microscopy test tomorrow to confirm.
*started competent cells for BAC with pBca1256-Bjh2344
*started transformations of competent cells BAC with pBca1256-Bjh2344, Bjh1934 with 4 PDD (2331, 2333, 2348, 2349. Added 10  μL of Mgs04 to rescue step. Added 30 μL by accident to Bjh2331 with 2344.
*did Eco Bam transfer of final PDD 2291 into 1601 KA vector. Plated that. (redo, forgot magnesium in rescue step.)
*poured Kam,Spec,Amp,Kan antibiotic plates.  
*checked controls for BAC with 1934 plates. Looked good.
[[Image:IMG_4958.JPG|100px]]negative control for amp.<br>
[[Image:IMG_4957.JPG|100px]]positive control for Cam/Spec<br>
[[Image:IMG_4956.JPG|100px]]negative control for Kan<br>


==To Do==
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, Sept 1 2010 (EDT)=  
*pick and grow up cells in TB(for assay)
*sequencing results for 7 looked good.
*miniprep TIm's Ecoli
*picked colonies to feed and assay on Thurs.
*transform pick 2291 and grow up to miniprep.  
*ran lysis assay in LB for 51, and 52. Saw clear lysis after about 2 hours. 52 seems to lyse a little sooner then 51.
*transform 2331,2333, 2348 2349 into 2343 w BAC, 2343, 1934 and 2344
*got two Pcon's from Tim/Sushant to put on the front of Lifeact. PCR off them with ca998 and g01001 and then Eco/Bam Eco/Bgl. Tape samples to the inside of TIm's fridge when done. WIll do tomorrow.  
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, 31 August 2010 (EDT)=
*miniprepped 3, 7, 8, and sent 7 in for sequencing with ca998.
*controls for "11" cells looked good
*picked cells to run preliminary lysis assay on Weds. before thursday


=[[User:Tahoura Samad|Tahoura Samad]] 16:09, 18 June 2010 (EDT)=
=[[User:Tahoura Samad|Tahoura Samad]] 18:36, 30 August 2010 (EDT)=
==PDD Subgroup Work==
*Did colony 2K for Pbad. Lifeact GFP. Band should be around 2000. Will miniprep, 3, 7, 8 tomorrow.  
*made competent cells of BAC with pBca1256-Bjh2344 in MG1655.
*Transformed 51, and 52 into "11" and JTK159 to replenish supply of PDD.  
1. added 500 μL of fresh culture to 50 mL of Spec/Cam  LB. Incubated for 3 hours in 37 shaker.<br>
*WIll assay on Wed, Thurs?
2. Chilled falcon tube.<br>
3. Lit flame, poured from small erlenmeyer flask into falcon tube. <br>
4. Chilled for 10 minutes.<br>
5.Chilled rotor for an hour.<br>
6. Spun down at 4100 rpm for 13 minutes. Make sure to refridgerate the centrifuge first. Temperature needs to be at 4 C. Make sure also to change the rotor program. <br>
7. Poured of supernatant.<br>
8. Resuspended in 2.5 mL of ice cold TSS. <br.
9. Distributed into 98 μL aliquots. <br>
----
*electroporated pB6 into MG1655 with pBca1256-Bjh2343.
Protocol:
Small Scale Electrocompetent Prep <br>
# Grow up strain to saturation
# Innoculate 2YT with 1:30 of o/n. (add antibiotic to 2yt, and add about 5 mL of 2YT. (5 microliters of antibiotic)
# Grow uninterrupted for 4hrs @ 30C for ubercompetency. (30 C is the room next to the -80 freezer.
# Cool tube/flask; transfer 1.5 mL to chilled epi tube.
# Spin down for 20 sec at 14.1 rpm, discard supe, place immediately on ice.
# Resuspend with .5 mL 10% ice cold glycerol.
# Spin down for 30 sec (14.1 rpm), discard supe, place immediately on ice.
# Resuspend with .5 mL 10% ice cold glycerol.
# Spin down for 30 sec (14.1 rpm), discard supe, place immediately on ice.
# Resuspend in a small volume (100 ul) 10% ice cold glycerol.
#Note: make sure to remove salts well, or else this won't work. You should dump of the supernatant, and than make sure its all gone by using a p200 to remove the little bit that's left.
# Add ~2.5uL  and 5 μLDNA to cells, pipette & stir to mix.
# Immediately add to chilled electroporation cuvette. (these have purple caps and are located in the cabinets) Add down the side so it is between the plates, and make sure there are no bubbles.
# Immediately shock. Electroporator is by the gel hut. remove the black slidy thing, wipe moisture off cuvette, and add so that notch in cuvette fits in groove in slide.
#stick it in the electroporator. voltage should be at 1600.
#press pulse twice. remove when Ch9 goes away.
# Immediately add 200 uL 2YT, pipette up and down to recover cells,
move to tube and keep warm. To do this, suck up 2yt in a pipette, and take it over with you. Add immediately.
# Put in shaking 37oC incubator for 1 hr
# Plate everything on appropriate antibiotic plate (allow to dry
before putting in 37oC incubator)
----
*started transducing Tim's Ecoli. Plated on Kan plates.
Transduction Protocol<br>
# Grow recipient strain overnight in LB medium (2 mL culture is plenty).
#On the next day, spin down 1mL of culture and resuspend in 1mL of
LB + 100 mM MgSO4 + 5 mM CaCl2 (basically, the P1 lysis mixture
without glucose, which is on the bench)<br>
For reference, that's:
  * 1 mL LB
  * 50 uL 2M MgCl2
  * 10 uL 500 mM CaCl2
(You can go 100 fold less concentrated and there will still be enough
recipient bacteria)<br>


3. mix 100 µL undiluted P1 lysate (from Josh's fridge)+ 100 µL recipient cells<br>
4. Incubate tubes at 37 ˚C for 30 min.<br>
5. Add 1mL of the 2YT/citrate mix, and incubate with shaking at 37 ˚C for 1 hr to allow
expression of the antibiotic resistance marker AND for Calcium
chelation, do not cut this short!)<br>
6. Spin down cells<br>
7. Resuspend each in 100µL 2YT/citrate.<br>
8.Prepare a plate spread with the selection antibiotic and 100 µL of 100 mM
citrate (pH 5.5) [do this by pipetting the 100mM citrate onto the top
of the plate, spread it around, and let dry].plate all of it on an
appropriate antibiotic-containing plate.
==To Do==
==To Do==
*On Monday, Eco/Bam Transfer the final PDD (2991)
*Miniprep 3, 7, 8 from col2k
*On Tuesday, pick and grow up. Miniprep Tuesday evening or Wednesday Morning
*pick colonies for assay on Wed. Either assay and invitro, or just invitro.  
*On Monday, start transformations of PDD into competent cells.
*Sequence pbad. lifeactGFP.  
*On Tuesday, pick cells and grow up.  
=[[User:Tahoura Samad|Tahoura Samad]] 19:29, 27 August 2010 (EDT)=
*On Monday, pick and grow up 2343 if they grew.
*Made JTK2801 competent cells "11". Will run controls when I transform on Monday.
*On Tuesday, make competent cells of Bjh2343 with BAC.  
*Make sure to email David Chen two days in advance about assaying.  
*On Monday, do something with Tim's Ecoli on plate
*On Monday, check controls for competent Bjh2344.  


=[[User:Tahoura Samad|Tahoura Samad]] 15:29, 17 June 2010 (EDT)=
=[[User:Tahoura Samad|Tahoura Samad]] 19:29, 26 August 2010 (EDT)=
==PDD Subgroup Work==
*Analyzed Tecan data Tim. 51 and 52, along with 41 lysed. Will hopefully will be continuing lysis assays with 51, 52.
*Checked on control for pB6 miniprep. (BAC with pBca1256-Bjh1934 cells in incubator) Was growth for 1:10 dilution. (1 colony), no growth for 1 μL and ~40 colonies for 3 μL, so it looks like the miniprep is fine.  
*Eco/Bam Eco/Bgl with Neb3 AraC. Pbad with Lifeact RFP.
[[Image:IMG_4892.JPG|100px]]BAC with pBca1256-Bjh1934 3 μL<br>
*picked up JTK8021 to make comp cells with and assay.
[[Image:IMG_4891.JPG|100px]]BAC with pBCA1256-Bjh1934 1 μL and 1:10 dilution 1μL. <br>
*picked Bjh2252 to miniprep and assemble with AraC.Pbad.
*BAC with pBca1256-2344 did not grow up well. It only had one colony (from glycerol stock) so we were concerned. Did colony PCR to check for BAC with oligos iGEM10_25, and iGEM10_26  and   am growing up.
==To Do==
*PCR looked good, with a band at 1 kb, which was the expected length for primers 25 and 26.  
*ReEcoBam 2271 into CA. First check with Tim though.
*[[Image:IMG_4894.JPG|100px]]
*Check with Tim about assaying when he's gone
*Picked ligation ecoli and grow up.'''Start time:10:40 am''' 
*Check with Tim what the heck to do with AraC. Pbad LIfeact
*make 2 1 L bottle of TB. TB is on the top shelf with the agar.'''Check back at 1:15 pm'''stored in cabinet by door e
*Make comp cells with JTK8021.


==To Do==
=[[User:Tahoura Samad|Tahoura Samad]] 19:29, 25 August 2010 (EDT)=
*On Monday, Eco/Bam Transfer the final PDD (2991)
*Ran lysis assay with 41, 42, 45-52 in JTK030, and in 159 3 different colonies. Will analyze Tecan Data tonight and tomorrow.
*On Tuesday, pick and grow up. Miniprep Tuesday evening or Wednesday Morning
*Talked to Tim about Lifeact parts. Lifeact GFP sequenced well but is not green. Abandon for now. LifeactRFP is heavily mutated in the Lifeact Region. Abandon
*On Monday, start transformations of PDD into competent cells.
*Will start assembly of Pbad. with LIfeact GFP and RFP. Grow up titration curve? Talk to Tim
*On Tuesday, pick cells and grow up.  
*Tomorrow: Make competent cells of BAC with pBca1256-Bjh2344.
*Tomorrow: Electroporate 2343 with BAC
*Tomorrow, miniprep 2348 and 2349
*tomorrow, do something with TIm's ecoli that he grew up.


=[[User:Tahoura Samad|Tahoura Samad]] 13:34, 16 June 2010 (EDT)=
=[[User:Tahoura Samad|Tahoura Samad]] 19:29, 23 August 2010 (EDT)=
==PDD Subgroup Work==
*2ab assembly products were not green and red. Will ask Daniela to pick on Tuesday to miniprep and see what is going on.
*check controls for competent cells for BAC with pBca1256-Bjh1934 in MG1655. Kan and Amp should be negative and Cam/Spec should be positive.  
*Neb2 Eco/Bgl LifeactRFP are now appearing red. Tim miniprepped them for me. Sent them in for sequencing to see what is going on.
*Kan and Amp were negative, Cam/spec was positive. looks good.
*Talked to Daniela and Tim about end of lysis assay on Friday. (everything in JTK159, plated on both Kan and Amp. Looks like just 51 and 52 lysed.
[[Image:SPEC.JPG|100px]] BAC with pBCA1256-Bjh1934 on Cam/Spec<br>
*Retransformed 41, 42, 45-52 into Jtk159 and Jtk030. Prepped blocks for Daniela to pick tomorrow so I can assay on Weds.
[[Image:AMP.JPG|100px]]BAC with pBCA1256-Bjh1934 on Amp<br>
*Picked BJh2541, Bjh2252 Eco/Bgl in Neb 2 and Bjh2251 by 2ab for Daniela to miniprep tomorrow.  
[[Image:KAN.JPG|100px]]BAC with pBCA1256-Bjh1934 on Kan<br>
*Also transformed Neb3 EcoBgl assembly products.


*check on transformations of pBca1256-Bjh2343 with pB6 (1:10 1 μL, 1μL, 3μL). Pick and grow up for competent cells and glycerol stock if there are any.
=[[User:Tahoura Samad|Tahoura Samad]] 19:29, 20 August 2010 (EDT)=
*none of these grew. at all.  
*Ran lysis assay in TB for 41, 42, 45-52. Left early to move in and will get results from Daniela.
*bad miniprep of pB6?
*check for this by transforming one of other competent cells that grew okay (pBca1256-Bjh1934 with with miniprep.)'''Plate at 12:50 pm'''
=[[User:Tahoura Samad|Tahoura Samad]] 16:35, 18 August 2010 (EDT)=
*repicked checked on transformations of MG1655 with pBca1601-Bjh2331,2333,2348,2349 and MC1061 with pBca1601-Bjh2331,2333,2348,2349 because I forgot to add magnesium. added magnesium.
==New Payload Work==
*Picked two colonies of each (16 total) and am growing them up in a 96 well block in the 37 C shaker. started at 12:15 pm. '''check on these at 5pm''
*Saw growth on plates, but no green and red colonies, so assembly was a failure.
*streaked glycerol stock of BAC with pBca1256-Bjh2344 to grow up tomorrow and make competent cells.
*Redid shortcut method of assembly, but digested EcoBgl with NEB3 instead of NEB 2.
*miniprepped PDD in MG1655 and MC1061-(2331, 2333, 2348, 2349)
*Also created new assembly trees and Eco Bammed everything into pmll vectors.
==Ligation Ecoli Work==
*Retransformed 41, 42, 45-52 and plated on AC for 41 and 42 and AK for 45-52 to avoid contamination issues. Asked Daniella to pick them for me tomorrow. Daniela said she would pick colonies for EcoBam stuff and do 2ab assembly on Friday. I'll do lysis testing.
*streaked glycerol stock of ecoli from Tim. Pick tomorrow.
=[[User:Tahoura Samad|Tahoura Samad]] 16:35, 17 August 2010 (EDT)=
==Additional Notes==
==New Payload Work==
*How to streak from glycerol stock:
*transformed everything from yesterday
1. dip tip of a p20 tip into glycerol stock<br>
==Biochipper's Device==
2. make 10 parallel slashes off to one side of the plate. Close tube and freeze it. Tim says its best if you actually do the plates right next to the minus 80 to keep the cells form thawing, because every time you thaw and refreeze, some cells die. <br>
*tried and failed at loading it. Maybe tape seal was no good?
3. take wire spreader and serially spread, flaming and ethanol between rounds.  
==Lysis==
==To Do==
*redid Amy's lysis assay in TB with no sucess. Amp plates seem to be contaminated and we plated on Amp. only. Will retransform 41, 42, 45-52 tomorrow.
*Check on AK stuff (MC1061, Mg1655 at 5:00 pm, 5:30 pm. Miniprep those guys tonight'''DONE'''
=[[User:Tahoura Samad|Tahoura Samad]] 16:35, 16 August 2010 (EDT)=
**Check on control for pB6 miniprep. (BAC with pBca1256-Bjh1934 cells in incubator)'''DONE'''
==New Payload Work==
**Pick 2344, and start growing up to make competent cells.
*retried Christoph and Conor's shortcut for assembly of the two new payloads by PCRing of PCON from Josh (plate 5, well c10) with phusion. Expected band length was 1000 bp. Cut and gel purified, and digested with Eco Bam. Also digested Lifeact GFP and RFP from Jin with Eco Bgl. Ligated Pcon eco bam into Eco/bgled lifeact rfp and gfp and transformed into Pir Lefty.
**Pick ligation ecoli and grow up.'''DONE'''
==Lysis Testing==
**make 2 1 L bottle of TB. TB is on the top shelf with the agar.'''DONE'''
*Amy ran lysis test and saw no lysis. Will rerun test for her in just TB tomorrow.
=[[User:Tahoura Samad|Tahoura Samad]] 16:35, 13 August 2010 (EDT=
*split choanos
*made JTK159 cells.
=[[User:Tahoura Samad|Tahoura Samad]] 16:35, 12 August 2010 (EDT)=
*Met up with Gayla and Tigran to learn how to make chips with Daniela. Practiced pouring PDMS, cutting devices, bonding etc. Make sure to get Gayla's guide.
*Found out that our jtk030 cells also have ptet in front of pir, which is leading to other problems. Tim grew up some JTK159, and we will make cells tomorrow.
=[[User:Tahoura Samad|Tahoura Samad]] 16:35, 11 August 2010 (EDT)=
*found out that all our payloads have pTet, which means they are useless with TetR. Need to make two new payloads. Abandoned assembly with new iBB because it includes pTet in front of GFP.
*Helped Amy wash samples for self-lysis test without payload.
*Ran TECAN along with macro scale test. Saw lysis for several constructs! See excel sheet.
*Also did stickiness assay to confirm lysis.


=[[User:Tahoura Samad|Tahoura Samad]] 16:16, 15 June 2010 (EDT)=
=[[User:Tahoura Samad|Tahoura Samad]] 16:35, 10 August 2010 (EDT)=
==PDD Subgroup Work==
[[Image:IMG_5844.JPG]]
*made glycerol stock of BAC with pBca1256-Bjh1934. See protocol online. Scanned on SimpleScan and logged in -80 stock on iGEM parts and data google doc.
=[[User:Tahoura Samad|Tahoura Samad]] 9:25, 9 August 2010 (EDT)=
----
*set up lysis assay. saw no lysis after 3 hours. grew upstairs in shaker.
*Started competent cells for BAC WITH pBca1256-Bjh1934 (Medium Scale)
*Choanos do not like to be left over the weekend.
1. added 500μL of overnight-grown culture to 50mL Spec/Cam LB in an Erlenmeyer flask<br>
*picked stuff for assembly final step.  
*put flask in shaker at 11:05 am. '''Check at 1:05 pm.'''
*cells were not really ready at 1:05, kept in incubator until 2:05 pm. 
2. checked overnight culture at 2:05 by spinning down 100 μL. Saw a pretty small white pellet.<br>
3. Transfered culture to a 50 mL falcon tube that was sitting on ice. <br>
4. Chilled overnight culture for 15 minutes to stop growth.<br>
5. Got chilled rotor from cold room. (was in there for a couple of hours.) Changed rotor and rotor program.<br>
6. Spun down cells at 4100 rpm for 13 minutes at 4 C. <br>
7. Poured of supernatant and resuspended in 2.5 mL of ice cold TSS.<br>
8. Made 25 98 μL aliquots in 0.5 mL eppendorfs on dry ice and put them in the same rack as our other competent cells in iGEM competent cell box 2.  the cells are labeled "5" and have been entered into the iGEM competent cell google doc. <br>
9. Streaked for contamination on Amp, Kan and Cam/Spec. Should grow on Cam/Spec, but not Amp, Kan.<br> '''Plate names: iGEM control for competent cells BAC with 1934'''
----
*Did genomic miniprep of Pb6.
Same steps as regular miniprep with the following changes. <br>
This procedure is identical to the normal miniprep procedure except
the step in red, and less culture is prepped<br>
1. Pellet 1.5 mL saturated culture by spinning full speed, 30 seconds.<br>
2. Add 250uL of P1 buffer into each tube. Resuspend the cells using<br>
a vortexer.<br>
3. Add 250uL of 2% SDS (stored in a 50 mL conicol) <br> (actually stored in falcon tube. Also, this step replaces the P2 step. <br>
4. Incubate at 55 degrees until clear (roughly 15-20min?). <br>
5. Continue as usual with N3 step. <br>
*stored in iGEM working box. '''tube name: g. pB6'''
*added 250 μL of 2% SDS after adding p1. Incubated at 55 until clear. '''check at 12:00 pm'''
----
*checked on transformations of MG1655 with pBca1601-Bjh2331,2333,2348,2349 and MC1061 with pBca1601-Bjh2331,2333,2348,2349.  
*MC1061 grew up really well, tons of really small colonies.  
*MG1655 had few, big colonies.  
1655-2331 (50-100 colonies) <br>
1655-2333-(35 colonies)<br>
1655-2348 (35-50 colonies)<br>
1655-2349(35-50 colonies) <br>
*Picked two colonies of each (16 total) and am growing them up in a 24 well block in the 37 C shaker.


==Gibson Work==
=[[User:Tahoura Samad|Tahoura Samad]] 18:32, 8 August 2010 (EDT)=
*diluted iGEM oligos 14-26 ( 5 μL oligo, 45 μL water)
*picked colonies for lysis assay on Monday and grew up in TB
*tested 1934 comp cells by transforming with 47.


==Additional Notes==
=[[User:Tahoura Samad|Tahoura Samad]] 11:25, 6 August 2010 (EDT)=
*iGEM competent cell box 2 is in the same rack as iGEM competent cell box 1, in the back column.
==Choano Assay==
==To Do==
*Set up lysis assay for 47 and 48 with payload in LB vs in ASW, with Mc1061 with and without ATC as a control. Results were that both control and positive ATC  OD dropped off at the same rate, possibly because the temperature control on the TECAN is at 26 degrees rather than 37.
*'''Important''' Check with Tim about the competent '''MG1655''' cells. According to TIm, in MG1655 cells, one or two point mutations make them Kan and Spec positive. Our cells are fine.'''DONE'''
*Lifeact RFP didn't grow up very well so was unable to get it to grow up well enough to make comp cells. Will retransform next week.
*check controls for competent cells for BAC with pBca1256-Bjh1934 in MG1655. Kan and Amp should be negative and Cam/Spec should be positive. '''DONE'''
=[[User:Tahoura Samad|Tahoura Samad]] 11:25, 5 August 2010 (EDT)=
*check on transformations of pBca1256-Bjh2343 with pB6 (1:10 1 μL, 1μL, 3μL). Pick and grow up for competent cells and glycerol stock if there are any. '''DONE'''
==Choano Assay==
*ask Tim what we should do with the MC1061 and '''MG1655''' with pBca1256-Bjh2331,2333,2348,2349 that are growing up in the 37 C shaker. '''REDO'''
*growth of cells in LB was poor, which confirms that life-time on each transformation is less than a week. Ideally within the first four days of life of the plate. Set up chonao assay again but saw no lysis. Will redo in TECAN tomorrow using TB to get better growth.  
*streak out ligation e-coli.
*made competent cells for Lifeact GFP in JTK030 and Bjh1934 in JTK030.
==Choanos==
*none of choano cultures, including 1:2 was at assaying density yet. Fed each 40 ul and let sit.


=[[User:Tahoura Samad|Tahoura Samad]] 14:53, 14 June 2010 (EDT)=
=[[User:Tahoura Samad|Tahoura Samad]] 9:32, 4 August 2010 (EDT)=
==PDD Subgroup Work==
==Choano Assay==
*Tim says the 10 μL BAC with pBca1256-Bjh2344 had two colonies, so he picked one, grew it up and made glycerol stock in the minus 80 for us.  
*Set up chonao assay. Washed saturated LB culture of 47 and 48 with payload delivery in choano media, ASW, TB and LB. Using 2 3 mL aliquots, added ATC to one tube and none to other to serve as control. DId not see lysis after 3 hours. Will grow up in LB and redo test on Thursday.
*picked pB6 so we can do a genomic miniprep on it and try to create BAC with pBca1256-Bjh2343 again.  
*re-cotransformed JTK030 with Bjh1934 and pBca1256 for assaying this week. Transformed JTK030 with lifeact GFP and RFP  and 1934to make comp cells.
*picked BAC with pBca1256-Bjh1934 3μL so we can create a glycerol stock and competent cells.  
==IBB==
*put both in shaker upstairs to incubate overnight. '''start time: 11:00 pm'''
*Eco Bammed RFP Boo15 out of hybrid plasmid into KC, and transformed into MC1061  pir righty.
*restarted Eco/Bam transfer of pBca1601-Bjh2331, 2333, 2348, 2349  into Shelley's AK 1601 because we forgot to add magnesium when we started the cloning steps on Friday.
*Quintara resequenced Rbs ibb, gfp and it looks perfect. Tim deleted the first three bp off the oligo, so that's why there is that deletion in the front of the rbs.
** To make I M magnesium sulfate
=Choanos==
-Added 12.4 grams of Magnesium sulfate to 50 ml of water in a falcon tube. Screwed on filter and vacuum filtered. <br>
*obtained a saturated choano culture. Split 1:2, 1:5, !;15 and fed 40 ul of choano smoothie. Will observe growth as compared to choanos in choano media.
*incubated at room temperature for 30 minutes: '''start time: 1:45'''
*transformed Eco/Bam transfer for all 4 parts into '''MG1655''' (4) and MC1061 cells. The MC1061s are in the -80 freezer, same row as usual, farthest left rack, second row, bottom box. They have a blue stripe. '''started transformation at 3:20''' '''should be done at 4:20'''added 10μL of MgS04.
*plated 400 μL of MgSo4 onto each of the Kan, Amp plates. Then plated 70 μL of each of the transformations. Set in 37 incubator.


==New project to get ColE2 and pir site into a strain of TIm's bacteria so we can combine the transformation and ligation step==
=[[User:Tahoura Samad|Tahoura Samad]] 13:32, 3 August 2010 (EDT)=
*get strain out of freezer. Its in row B8. In -80 freezer, 2nd row from the top leftmost rack, in blue bottomed box.
[[Image:IMG_5767.JPG]]
[[Image:IMG_5775.JPG]]
[[Image:IMG_5736.JPG]]
==Choano Assay==
*ran assay of taking pictures of choanos eating just payload bacteria every five minutes for an hour. Saw photobleaching at a round minute 82. Arrived at scope 15 minutes after having fed choanos and were not seeing any rod phenotype. Hard to conclude the details of choano digestion because choanos keep moving slightly in and out of plain, messing up focus of FPs. Need to figure out how to fix choanos in space.
*miniprepped Rfp terminator and GFP rbs ibb. RFP terminator mapped strangely. Its a hybrid plasmid. Solution: PCR off hybrid plamsid using Ca998, G0100, digest, and ligate into correct plasmid.
==Lysis Assay==
*Did a loose assay to check if construct 47 and 48  without payload were lysing in ASW. Grew up in LB to saturation, washed and added ATC. Did not lyse. Picked and grew up colonies in TB to redo assay tomorrow. Will use just TB culture as a control, because it should lyse obviously within two hours according to Amy.
=
=[[User:Tahoura Samad|Tahoura Samad]] 16:03, 2 August 2010 (EDT)=
==Chonao Assay==
*Checked 3 day culture of flavobacterium free chonaos. Not as dense as a 3 day culture should be.
*Ran assay of TetR 47, TetR48 at +15, 0 -15 and -30. After 1 hour, seeing some rod shaped bacteria, but some empty choanos. Didn't see desired phenotype after 1 or 4 hours. Images are clearer without biofilms though.
*Surprised not to see some of landmarks of digestion we expected to see. Rounding up? Run 1 hour assay tomorrow to establish baseline
==Other==
*streaked out JTK030. Pick to make comp. cells tomorrow. Make iGEM glycerol stock of Jtk030.  
==Lifeact==
*picked LIfeact GFP to make comp cells today. In JTK030.
*picked LIfeact RFP in 1256


==Additional Notes==
=[[User:Tahoura Samad|Tahoura Samad]] 16:03, 29 July 2010 (EDT)=
==Choano Assay==
*began set up for choano assay at 10:00.
*used whole mLs for each of three hour time points because of loss of choanos during washing.
# 10:15 am, made 3 1 ml aliquots of each TetR 47 and TetR 48. TetR 47 did not grow up as well as TetR 48.
# 10:30 added ATC to samples with ATC for 90 minutes. (1 μL per mL of bacteria, keep in shaker.)
# 10:45 split choanos 1:15.
#11:00 Added ATC to samples with ATC for 60 minutes. Started filtering choanos
#11:30 Added ATC to samples with ATC for 30 minutes.
#11:40 Added NH4CL to samples that needed it.
#12:00 Fed choanos. Took about 10 minutes.
#12:35 Washed choanos with 1 mL of choano media and chased with 25 mL of white ecoli (plain MG1655)
#1:00 Started filtering choanos
*looked at chonaos at around the two hour mark. Very little fluorescence at all, only getting fluoresence when we bumped up the exposure to like 500 ms. Possible that the cells are lysing way before they are eaten. Chris says to try intervals between 15 minutes before to 15 minutes after feeding.
*in assay, chase with less white ecoli.
*from assay, can conclude that choanos complete digestion definitely within 2 hours, probably within 1.
*no such thing as narchoanos. :( just choanos with two flagella.
==IBB Work==
*miniprepped 3 of 4 colonies that weren't cotransformed for assembly without ATP and with DNTPs.
*picked and grew up Bca1092
==To Do==
==To Do==
*check on pBca1256-Bkh1934 and pB6.
*streak out JTK030 from somewhere
*Make glycerol stock
*transform 1934 into JTK030 and make into competent cells.
*Make competent cells of pBca1256-Bjh1934
*transform 2255 into JTK030 and make into competent cells.
*Genomic miniprep pB6
*EcoBam 2254 again and transform into JTK030.
*transform and plate pBca1256-Bjh2343 in MG1655(1) with pB6
*redo assembly of GFP RBS ibb.
*miniprep BCA1092 and do assembly of RFP terminator
*pick and grow up 47 to miniprep, 48
*miniprep 1934 payload.
=[[User:Tahoura Samad|Tahoura Samad]] 15:12, 28 July 2010 (EDT)=


=[[User:Tahoura Samad|Tahoura Samad]] 13:12, 11 June 2010 (EDT)=
=[[User:Tahoura Samad|Tahoura Samad]] 15:12, 27 July 2010 (EDT)=
==PDD Subgroup Work==
==IBB Work==
[[Image:IMG 4816.JPG|100px]]PbCA1256-bJH2344 with 1 μL<br>
*miniprepped RFP KA and mapped it to make sure it wasn't methylated at bgl and bam.
[[Image:IMG 4817.JPG|100px]]PbCA1256-bJH2344 with 3 μL<br>
[[Image:IMG_5567.JPG|100px]]eco bam of rfp bam xho bgl xho. looks good
[[Image:IMG 4815.JPG|100px]]PbCA1256-bJH2343 with 3 μL<br>
*got BCA1092 from Jin and transformed it into righty.
[[Image:IMG 4814.JPG|100px]]PbCA1256-bJH2343 with 1 μL<br>
*miniprepped TIm's GFP.
*started assembly of AC Tim's GFP with CK rbs ibb.
*sent RBS ibb in for sequencing.
==Lifeact==
*Lifeact RFP transformations came up green, which means i used the wrong DNA. Redid Eco Bam and transformed into MG1655.  
==Choano Assay==
*had Conor plate my transformations, but I'm worried about them actually being co transformed because top plated spec and you can see that they are all growing up around the edges, where there might not be spec. Fishy. Redid cotransformations with 1 ul and 3 ul of straight DNA of each.


*checked on second round of BAC with pBca1256-Bjh2343, BAC with pBca1256-Bjh2344, and BAC with pBca1256-Bjh1934 plates tomorrow. No growth for  BAC with pBca1256-Bjh2343, BAC with pBca1256-Bjh2344 (1 μL and 3 μL) but ~50 colonies on pBca1256-Bjh1934: 1 μL and decent growth for pc.
=[[User:Tahoura Samad|Tahoura Samad]] 13:11, 26 July 2010 (EDT)=
*Talked to Tim. Tim says since BAC is 40 kb, not 15 kb, the miniprep we used is not really good, so I'll re-miniprep BAC on Monday.  
==Choano Assay==
*In the mean time, I retransformed BAC with pBca1256-Bjh2343, and BAC with pBca1256-Bjh2344 and added 10 μL of '''undiluted''' pB6 miniprep.'''Incubation start time 11:04pm'''
*TetR 47 and 48 didn't grow up because I transformed them into cells without pir. They are not in PMLL, but in entry vectors. Redid those transformations today. (cotransformation)
*plated entire transformation on Cam Spec plates.  
*filtered chonaos, fed with 1 μL of overnight culture, waited 20 minutes, washed and then chased with 15 μL of white bacteria.  
*Eco/Bam transfered 1601CA Bjh 2333 L, 1601CA Bjh2348 L, 1601CA Bjh2349, and 1601CA Bjh 2331 in pBca1601 AK. We did this because BAC is CAM resistant, so if both parts are Cam resistant, it is possible to lose BAC, which is only single, and not be able to tell. So in the end, we will end up quadruple plating our stuff.
*observed after 1 hour, 4 hours. Still too much green bacteria outside cells, looks like they are still eating. Lots of rod-shaped. Strangely, not a lot of degradation of gfp after 1hour in cells without nh4.  
==Creating Competent Lefty, Righty Cells, (MC1061 pir 1)==
*lifeact: after 1 hour, cells are green, can see rod shaped bacteria. After four hours, choanos look pretty empty (digested?) Interesting to note that several times we observed choanos with extremely round dot of green adjacent to them? Also saw balls of green in their bodies. Actin is balling up?
==IBB==
*did Eco/Bam of Lifeact RFP into 1256 (f4 on TIm's stock plate 1) background will be white. Will transform tomorrow.
*picked and


*created glycerol stock for  MC1061 pir1 lefty and righty cells. Logged on google doc and put into -80 freezer
*helped Tim a little to make competent lefty righty cells.


=[[User:Tahoura Samad|Tahoura Samad]] 16:56, 9 June 2010 (EDT)=
=[[User:Tahoura Samad|Tahoura Samad]] 13:11, 22 July 2010 (EDT)=
==PDD Subgroup Work==
==PDD Subgroup Work==
*checked controls for competent  cells. They looked good with no growth on Amp, Cam and Kan and growth on Spec.
*test for competency of 3s was interesting. Cells not as good as original with 25 colonies for 2349 and about 50 for 2333. Might remake cells. Probably let them sit out for too long.
[[Image:NegControlAmp.jpg|200px]] Negative Control for Amp<br>
==IBB==
[[Image:NegControlKan.jpg|200px]] Negative Control for Kan <br>
*digested rbs. ibb and gel purified product. (small fragment)
[[Image:NegControlCam.jpg|200px]]Negative Control for Cam<br>
==Lifeact==
[[Image:NegControlSpec.jpg|200px]]Positive Control for Spec <br>
*transformations worked. Will pick 2255 on Sunday, make competent cells on Monday.  
*checked on transformations with BAC. All failed. 0 colonies for both plates. Possible reasons for failure: We added twice as much KCM as we should have. Also, Tim said some BACs are just hard to transform.  
==To Do==
[[Image:BAC2343.jpg|200px]] Transformation of pBca1256-Bjh2343 in MG1655 with BAC <br>
*Pick 3s, Bjh2255 on Sunday.  
[[Image:BAC2344&1934.jpg|200px]] Transformation of pBca1256-Bjh2344 in MG1655 with BAC (left) ;Transformation of pBca1256-Bjh1934 in MG1655 with BAC (right) <br>
 
*threw plates away in red garbage
=[[User:Tahoura Samad|Tahoura Samad]] 12:50, 21 July 2010 (EDT)=
*redid transformations with 1μL, and 3 μL of '''undiluted''' pB6 miniprep. Scaled down KCM to 15μL because our cell aliquots are only 98 μL '''Start time in incubator: 11:08 am.'''
*Lifeact assembly failed again. Found out that Jin has GFP and RFP versions of the part I'm trying to assemble. Abandoned LIfeact Assembly.
*plated all 159ish μL of each transformation on Spec, Cam plates (blue yellow). '''plate names: 1 μL BAC with pBca1256-Bjh2343. 3 μL BAC with pBca1256-Bjh2343. 1 μL BAC with pBca1256-Bjh2344 3 μL BAC with pBca1256-Bjh2344, 1 μL BAC with pBca1256-Bjh1934, 3 μL BAC with pBca1256-Bjh1934.'''
[[Image:IMG_5467.JPG|100px]]bgl/xho <br>
[[Image:IMG_5472.JPG|100px]]bam xho, eco, xho <br>


==Assembly of iGEM parts 001 and 009==
*Got Lifeact RFP and Lifeact GFP from Jin. Transformed Lifeact RFP into MC1061 and Lifeact GFP (Bjh2255)into MG1655. Lifeact RFP 2254 needs to be ecobam transfered into pbca1256.
*started miniprep of assembly 2 with Amy
*gel purify PCR product, digest and ligate into vector, retransform. Digestion done at 11:06 pm. Gel came up empty, so redid PCR wit 1:10 dilution of olios.
*cut out bands from Amy's gel. Lane 1: 1L (desired length: about 1100) Lane 2: 1R (~1300) Lane 3: 9L (~1200) Lane 4: 9R (~1500)  
*do test transformation with 3s to make sure they work equally well as the last ones with 2333 and 2349.
*the point of the gel purification  was to get rid of parent vector and to select for the side of the vector we want,  
*transformed Amy's new constructs into 3s. Set up choano assay for Monday.
*the part lengths were obtained from the part calculator sequences and the lengths of the digested pieces that would ultimately be included in the iGEM10_001 or 009 composite part after ligation and appropriate antibiotics selection.
*Get parts from Jin
I combined the 1L with the 1R and the 9L with the 9R gel fragments and performed a Zymo Gel Purification, eluting with 17uL of water for each.
*Figure out 1934 payload restructure.
----
*controls for competent cells looked good, with no growth on Cam, Kan and Amp and growth on LB and Spec.
*Ligation:
[[Image:IMG_5477.JPG|100px]]LB <br>
*Set up ligation mastermix
[[Image:IMG_5476.JPG|100px]]Cam <br>
Mastermix <br>
[[Image:IMG_5475.JPG|100px]]Amp <br>
6.5uL ddH2O<br>
[[Image:IMG_5474.JPG|100px]]Kan <br>
1uL T4 DNA Ligase Buffer (black-striped tubes)<br>
[[Image:IMG_5473.JPG|100px]]Spec <br>
0.5uL T4 DNA Ligase<br>


DNA
6uL of L+R gel purified DNA<br>
Added DNA to MM at 3:54pm. The ligation will be done at 4:30pm.<br>
----
==Assembly with Gibson Oligos==
*Conor showed me how to design Gibson Oligos using the Gibson Oligo Spreadsheet
*Designed the Gibson Oligos for iGEM10_20 and checked Conor's oligos for iGEM10_13.
**Note: 20 mers can be longer than 20 so that Tm is at least 55. Additionally, oligos need to end on a g or a c.


==Additional Notes==
==To Do==
==To Do==
*check on BAC with pBca1256-Bjh2343, BAC with pBca1256-Bjh2344, and BAC with pBca1256-Bjh1934 plates tomorrow.
*transform some of JIn's stuff into cells. Pick, grow up, miniprep. Eco Bam 2254them into 1256.
*pick colonies and grow up to make BAC -80 freezer stock
*re _eco bam transfer RFP!
*digest, ligate etc rbs.ibb and send for sequencing.
 
=[[User:Tahoura Samad|Tahoura Samad]] 17:03, 20 July 2010 (EDT)=
*Lifeact failed to assemble.
*made competent 3 cells
*talked to Chris about choanos test with just GFP.
*Redid Lifeact assembly.


=[[User:Tahoura Samad|Tahoura Samad]] 17:03, 19 July 2010 (EDT)=
*Assembly of LIfeact part
*set up FACs training.
=[[User:Tahoura Samad|Tahoura Samad]] 17:03, 16 July 2010 (EDT)=
==PDD Work==
*did transformations of 3s with PDD
*did transformations of MG1655 with Tim's RFP (a5, plate 2). Could plate that after heatshocking because its amp.
*streaked out 3s and 5s
==To Do==
*Saturday: pick and grow up 3s,Mg1655 with RFP.
*Sunday: assay. Split choanos 1:5 so that we have some for monday.
*Sunday:pick and grow up RFPs so that we have samples to look at under flow cytometer.
*Monday: split choanos 1:15


=[[User:Tahoura Samad|Tahoura Samad]] 16:46, 15 July 2010 (EDT)=
==PDD Subgroup Work==
==PDD Subgroup Work==
*Created -80 Stocks for pBca1256-Bjh1934 in MG1655, pB6 in DH5-alpha, MG1655, Bjh2343 in MG1655, Bjh2344 in MG1655r  using [http://openwetware.org/wiki/-80_Glycerol_Stocks this protocol]
*assembly of LifeAct abandoned. Now up to Christoph and Daniela
*scanned and logged these stocks as the first iGEM -80 stocks (created a stock box for -80). Scanner is located next to pipetting robot. Spreadsheet for -80 stock is located on 140L parts spreadsheet on google docs.
*learned how to use microscope at 10 am (Arkin Lab). Looked at choanos after 1 hour and got pictures
----
*set up time scale for weekend.
*Did Miniprep of PB6 starting with 2mL of the overnight culture. Stored eluent (50 μL) of DNA in iGEM working box.
==IBB==
-----
*miniprepped IBB in CK Righty and CA Righty. Will not sequence.  
*Made Competent Cells for pBca1256-Bjh1934 in MG1655,Bjh2343 in MG1655 and Bjh2344 in MG1655:
*go over assembly tree of that with TIm.
Recipe<br>
*got rbs oligo and resuspended at 100 mM (nm times 10 μL.)
1. added 500μL of overnight-grown culture to 50mL Spec LB in an Erlenmeyer flask
for MG1655: (1:100)<br>
2. added 1mL of overnight-grown culture to 50mL LB (no AB) in an Erlenmeyer flask. For this one, we doubled the amount of cell culture because we added glycerol to our cells, so they were half the dilution they should have been.<br>
3. Put both flasks put on shaker in lab at 12:00<br>
4. Took cells out of shaker. Had to put the MG1655 back in for longer because they grew very poorly. Probably because we added glycerol. <br>
5. Followed protocol for large scale competent cells with the following amendments:
*spun cells down for 13 minutes at 4100 RPM.''' MAKE SURE TO BALANCE THE CENTRIFUGE to within 1 gram.!!!!'''
6. While centrifuge was running, we got .5 ml eppendorfs and labeled them 1, 2, 3, 4, while keeping them on dry ice. Dry ice is so the cells flash freeze, which is good if you are working slowly.
The numbers 1, 2, 3, 4 correspond to different competent cells, and the key can be found in the iGEM parts and data spreadsheet on google docs.<br>
7. Removed falcon tubes from centrifuge and poured off supernatant into a bottle in the sink. Add bleach etc. <br>
8. Resuspended pellet by vortexing in 2.5 mL of TSS, which can be found in the plate fridege, top right back.<br>
9. Put on ice, and aliquoted 98 μL in 25 Eppendorfs for each of the four. <br>
10. Put in -80 freezer. Third shelf? (or same shelf as JTK049, rack furthest to the rights.<br>
11. Set up positive and negative controls for our competent cells<br>
*Streaked cells on Cam, Amp, Kan and Spec. Cam, Amp, and Kan should come up negative for 1, 2, 3. Cam, Amp, Kan and Spec should for 4.<br>
----
*Transformed BAC into 1, 2, 3.
*Plated on Cam, Spec plates.


==To Do==
==To Do==
*check controls for competent cells. Kan, Cam and Amp should be negative (no cells) spec should be positive. Exception: 4 (MG1655) should be negative for all 4 plates. '''DONE'''
*transformations of 3s with PDDs
*make competent cells for pBca1256-Bjh2343 with BAC, pBca1256-Bjh2344 with BAC, pBca1256-Bjh1934 with Bac.
*transformation of RFP into Mg1655
*Find out what BAC (PB6) is
*start PCR of rbs onto IBB.
*Look up what pBca1256-Bjh1934 is. (short description, part name)
*split choanos so i have 50 mls on Sunday.
*streak out 3s and 5s
 
=[[User:Tahoura Samad|Tahoura Samad]] 13:48, 14 July 2010 (EDT)=
==PDD Subgroup Work==
*picked and grew up 3 series for microscopy
*picked and grew up 3s and 5s for minimal media test. Same procedure as last time, but no duplicates, and grow up in minimal media with 10 mM MgSo4 overnight for 16 hours.
 
==Additional==
*Checked controls for Pir1 MC1061 lefty and rightys. Controls looked good.
==IBB Work==
*Miniprepped Ptet and b1006 for assembly trees
*Checked with TIm how to make oligo to put rbs on the front of ibb and made oligo. Temo of annealing region should be 55 c. THe nonsense sequence in front of the rbs is just random stuff Tim put there.
==Lifeact==
*miniprepped lifeact that is actually KA and mapped to make sure it was correct. Expected sizes 1300, 1700
[[Image:IMG_5353.JPG|100px]]
[[Image:IMG_5355.JPG]]ca ka ck
 
.


==Additional Notes==
=To Do=
*shaker on fourth floor is not at exactly 37, closer to 33, 34, so things take longer
*Jin wants to redo the minimal media test, but not in duplicate and make two aliquots after filtering the choanos.  
*make sure to scale down KCM by half for all the competent cells we made. (1, 2, 3, and 4)
*Pick 3 and 5 on Tuesday
*BAC stands for Bacterial Artificial Chromosome.
*Split chonaos 1:15 on thursday again
*Pick and grow up CK Righty, CA Righty,RFP! and 3s.


=[[User:Tahoura Samad|Tahoura Samad]] 13:46, 8 June 2010 (EDT)=
 
=[[User:Tahoura Samad|Tahoura Samad]] 13:56, 13 July 2010 (EDT)=
==PDD Subgroup Work==
==PDD Subgroup Work==
*all five transformations grew up well. The 2343 and 2344 appeared red.  
*started Eco Bam transfer of 1882 part for lifeact tree into AK.
* Picked colonies for 2343(spec), 2344(spec), 1256-1934(spec), pb6(Cam) and MG1655 (LB) in a 24 well plate. Put in 37 C shaker. '''Check on them at 4 pm.'''
*sent Ptet+Lifeact in for sequencing. We suspect it is bad because one of the inputs was in KA, which was actually AK.
==Manual Assembly of iGEM10_13 and iGEM10_20==
*Talked over results of minimal media testing with Jin. Results were ambiguous because there was inconstancies between the duplicates. Additionally, Jin thinks that Mg in the choano media may be obscuring the results. One thing he observed that he wants to investigate further is that he thinks 37 C might be slowing down the choano's digestion.
*began manual assembly of assembly tree for iGEM10_13 and iGEM_20. (create iGEM10_008, 009, 014, 015, 016.)
*Split chonaos 1:2 so they will be ready for assaying on Thursday
*set up Lefty and Righty digests and incubated: '''incubation should be done at 2:50 pm'''
*Started transformations of 3s for practice assay on Thursday Morning. Talked to Jin. He said transformations are good for about 3 days on plate.  
**Digested 8+4, 7+11 (x2), 9+20 (x2) with Lefty MasterMix.  
*transformed CA IBB eco bam transfer into Righty cells and CK IBB DNA (old) into righty cells.
**Digested 9+3, 8+1, 7+2, 7+19, 8+12 with Righty MasterMix.
==Additional==
*Zymoed the 8 digests.
*Checked controls for Pir1 MC1061 lefty and rightys. Controls looked good.
----
[[Image:IMG_5306.JPG|100px]]Kan <br>
*Set up ligation reactions;
[[Image:IMG_5307.JPG|100px]]Spec <br>
*Ligate
[[Image:IMG_5308.JPG|100px]]Cam <br>
**Followed changed protocol (see 6/7/2010):  
[[Image:IMG_5309.JPG|100px]]Amp <br>
**(Amy)Set up 5 ligations (made MasterMix recipe x6) 
[[Image:IMG_5310.JPG|100px]]LB <br>


Mastermix<br>
==IBB Work==
6.5uL ddH2O<br>
*transformed CA IBB eco bam transfer into Righty cells and CK IBB DNA (old) into righty cells.
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)<br>
*Made assembly trees of parts with Tim.
0.5uL T4 DNA Ligase<br>
*picked some of Ptet and bl006 of Christoph's plates for my assembly.  


DNA<br>
=To Do=
3uL Lefty vector<br>
*Jin wants to redo the minimal media test, but not in duplicate and make two aliquots after filtering the choanos.  
3uL Right vector <br>
*Pick 3 and 5 on Tuesday
Start time ''(4:15 pm, End time 4:45 pm)'
*Split chonaos 1:15 on thursday again
----
*Pick and grow up CK Righty, CA Righty,RFP! and 3s.
*transformed ligation into Jtk049 (not methylated, from -80 freezer) because there were no lefty and righty cells left.  
*plated and put in incubator
**iGEM10_016, 008 on CK
**iGEM10_015, 009 on AC
**iGEM10_014 on KA


==Manual Assembly of iGEM10_001, 002, 003==
*Ran gel of colony PCR
*Parts looked correct.


==Additional Notes==
*Dishwashing room: on 4th floor. Can fit 15 blocks in a rack. Dishwasher on right is messed up.


==[[User:Tahoura Samad|Tahoura Samad]] 17:20, 12 July 2010 (EDT)==
[[Image:IMG_5276.JPG|200px]]
*ran 1B of colony pcr for life act again. looked good. will send for sequencing tomorrow
*ran Jin's minimal media assay
*Eco Bam transfered Ck lefty into Ca. will transform into righty cells.
==[[User:Tahoura Samad|Tahoura Samad]] 17:20, 9 July 2010 (EDT)==
*miniprepped col PCR results, which grew up equally poorly. May have a hit on the first one. Will transform on Monday
[[Image:IMG_5247.JPG|100px]]
*did transformations of 3s with 5 pdds.
*set up wells so amy can pick on Saturday
=To Do=
*Sunday: split choanos 1:5 so i have enough for Monday's testing. Set up assay for Jin, filter after 2hours. Pick and grow up 3s and 5s for 16 hrs for minimal media testing
*pick Pir1 lefty and righty for competent cell prep.
=[[User:Tahoura Samad|Tahoura Samad]] 15:18, 8 July 2010 (EDT)=
==PDD Subgroup Work==
*direct Jin to confirm which of the combinations is actually working.
*remind Tim to email Jin about scope.
==Life Act==
*Even after 24 hours, the bacteria from the colony PCR still did not grow up well. Only saw growth for a couple. Miniprepped colonies 6, 9, 19, 20. Restriction mapped Bam, Eco, excpected band length of 167 and 2700
[[Image:IMG_5213.JPG|500px]]
[[Image:IMG_5235.JPG|500px]]
==Other==
*picked MC1061 pir1 lefty and righty and am growing up in LB.
=[[User:Tahoura Samad|Tahoura Samad]] 16:10, 7 July 2010 (EDT)=
==PDD Subgroup Work==
*nothing
==LifeAct==
*redid colony PCR with 24 new colonies. Results looked good, with many bands at expected length of 357.
[[Image:IMG_5183.JPG|100px]]
*will miniprep in the afternoon.
==Other==
*streaked out MC1061 pir lefty and righty since we're almost out. Will make competent cells on Friday.
==To Do==
==To Do==
*create glycerol stock of pb6 for -80 freezer. '''DONE'''
*Pick and grow up Pir Lefty and Righty for competent cell prep on Friday.
*Create -80 stock for all 5 and competent cells for everything but pB6.  
*On Friday, do transformations of all 3s with all five PDDs.
*coat hangers? '''DONE'''
*On Friday, make competent cells.
*On Saturday, take out of incubator, pick and grow up.
*On Sunday, feed to choanos.
*On Sunday, pick and grow up 3 and 5 for Monday.
*ask Tim what to do with IBB.
*get solutions of MMM and MM0 from Jin.
*ask Jin #grow up in shaker for 2 more hours. (Ask Jin what how much of each solution to grow them up in.)
*split choanos 1:5 on Sunday, so we'll have enough for Monday. Check with conor how to get 17 mL of choanos for Monday.
 
=[[User:Tahoura Samad|Tahoura Samad]] 18:16, 6 July 2010 (EDT)=
==PDD Subgroup Work/Discussion With Jin==
*talked to Jin. He was not able to look at the combinations from Friday. :(
*Jin says to let all the different combinations rest, and focus on the 1934 payload.
*Jin also says to start the minimal media testing to see if pb6 works.
------
Minimal Media Testing
*Day 1
#Streak out 3, 5
#Pick and grow up in TB with 30 mM MgS04 (30 μL per mL) overnight.(16 hrs) Do two colonies of each 3 and 5.
*Day 2
#Dilute 100 fold by adding 30 μL of overnight culture to 3 mL of TB. (4 tubes, 2 of 3 and two of 5)
#Grow for 2-3 hours more. (2 hours)
#make 2. 1 mL aliquots of 3 and 5. (Four of each) (8 tubes total)
#Wash 3 times.
## Wash two of each with minimal media with magnesium (MMM) and Minimal media without magnesium. (MM0) Get these solutions from Jin.
##When you wash these guys, spin the cells down, carefully pipette off the supernatant without touching the pellet, resuspend with solution etc .3 times.
#grow up in shaker for 2 more hours. (Ask Jin what how much of each solution to grow them up in.)
#filter 17 mL of choanos
#incubate 8 mL of choanos with 10 μL per mL (10 mM ) NH4Cl for 10 minutes.
# feed 10 μL of bacteria to choanos. Look at after 5 hours.


=[[User:Tahoura Samad|Tahoura Samad]] 14:15, 7 June 2010 (EDT)=
==PDD Subgroup Work==
referenced Jin's notes:[[Image:jin_Notes1.JPG|200px]]
*(with Conor) Transformed Mg1655 cells (from -80 freezer) from Jin with two PDD 2343, 2344( from freezer box).
**Diluted DNA by 10x ( 9 μL dH20, 1  DNA), added 1 μL to cells.<br>
* pB6 (BAC) see image, arrived from Germany. Wear gloves because its BSL 2. Currently in fridge. TIm streaked it on a plate. (Note: Streak such that the concentration is reduced as you go along.) The part we need arrived in bacteria, so we need to grow them up and miniprep them to get the DNA we need.
* Transformed MG1655 cells with pBca1256-Bjh1934 (from Jin's Overflow box 1). Tube name: ''1256-1934 into MG1655''
* Plated our transformations (2343 in MG1655 and 2344 in 1655) on Spec plates (yellow). Plate names: ''2343 into MG1655, 2344 into MG1655''
* Created spreadsheets for iGEM Freezer boxes
* Tried to enter part information into Clotho, but were unable to save in FlatData
*entered parts into Google Group spreadsheet
*plated transformation of MG1655 with pBca1256-Bjh1934. Plate name: ''1256-1934 into MG1655''


== Manual Assembly of iGEM10_003==
*Digestion
**prepared Righty Master Mix (see group lab notebook)
-----
Lefty Mastermix Recipe (for 4uL miniprep DNA) <br>
4uL water<br>
1uL of NEB2<br>
0.5uL XhoI<br>
0.5uL BamH1<br>


Righty MasterMix (for 4uL miniprep DNA)<br>
==Lifeact==
4uL water<br>
*ran colony PCR for 32 colonies.
1uL of NEB2<br>
[[Image:IMG_5167.JPG|1000px]]
0.5uL XhoI<br>
*minipreped 30 and 32, but they turned out to be contransformed. Of 32 colonies, 7 were contransformed.
0.5uL BglII<br>


*R masterimix  added to 7+23, 8+6, 7+5
==NLS==
*L mastermix added to 9+8, 7+11, 9+20
*sequencing results looked okay.
----
*iGEM_028, CK lefty looks perfect except for a point mutation in the EcoR1 site. Looking at the abi, i think it might be a miscall.
*Ligation:
*iGEM_029, CK Righty looks perfect except for a one base deletion after the Pts1 site. Looking at the Abi file, exactly at this point, the sample gets mixed reads. ?
*iGEM_030, CA Righty looks perfect except for the point mutation in the Ecor1 site. Looking at the abi, i think it might be a miscall.


Normal protocol with slight changes: <br>
==To Do==
Mastermix Recipe<br>
*On Friday, do transformations of all 3s with all five PDDs.
6.5uL ddH2O<br>
*On Saturday, take out of incubator, pick and grow up.
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)<br>
*On Sunday, feed to choanos.
0.5uL T4 DNA Ligase<br>
*On Sunday, pick and grow up 3 and 5 for Monday.


DNA Recipe<br>
3uL Lefty vector<br>
3uL Right vector


*Incubated on bench until 5:37pm. Does not need to sit for full 10 minutes on ice. Tim says one or two minutes should be fine. Heat shock for 90s.
=[[User:Tahoura Samad|Tahoura Samad]] 13:22, 5 July 2010 (EDT)=
* Transformed 9+20,7+5 (iGEM10_003) and 9+8, 7+23 (iGEM10_001) into the Righty strain. And 7+11, 8+6 (iGEM10_002)into the Lefty strain. Gave to Tim to plate.
*picked and grew up 1, 2, 6, 7, to make competent cells.


==Additional Notes==
=[[User:Tahoura Samad|Tahoura Samad]] 13:22, 2 July 2010 (EDT)=
*Zymo can be used to of get rid of restriction enzymes. In this case, we can't heat kill because BamH1 survives.  
*most of my transformations with the new competent cells were successful.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Competent cell'''
| align="center" style="background:#f0f0f0;"|'''Number'''
| align="center" style="background:#f0f0f0;"|'''PDD'''
| align="center" style="background:#f0f0f0;"|'''Growth in initial competent cells'''
| align="center" style="background:#f0f0f0;"|'''Growth in Small Scale Competent Cells'''
| align="center" style="background:#f0f0f0;"|'''Result of Assay with Jin'''
| align="center" style="background:#f0f0f0;"|'''''
| align="center" style="background:#f0f0f0;"|'''Conclusion'''
|-
| pBca156-Bjh2343||1||2331||0||tons of colonies||NOT DONE||||transform new, assay
|-
| pBca156-Bjh2343||1||2333||0||tons of colonies||NOT DONE||||transform new, assay
|-
| pBca156-Bjh2343||1||2348||1 colony (twice)||tons of colonies||NOT DONE||||transform new, assay
|-
| pBca156-Bjh2343||1||2349||0||50-100 colonies||NOT DONE||||transform new, assay
|-
| pBca156-Bjh2343||1||2291||0||100-200 colonies||NOT DONE||||transform new, assay
|-
| pBca156-Bjh2343 with BAC||7||2331||0||15 colonies||NOT DONE||||Today
|-
| pBca156-Bjh2343 with BAC||7||2333||0||2 colonies||NOT DONE||||Today
|-
| pBca156-Bjh2343 with BAC||7||2348||0||4 colonies||NOT DONE||||Today
|-
| pBca156-Bjh2343 with BAC||7||2349||0||0||DEAD||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh2343 with BAC||7||2291||0||0||DEAD||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh2344||2||2331||0||tons of colonies||NOT DONE||||make new, transform, assay
|-
| pBca156-Bjh2344||2||2333||0||tons of colonies||NOT DONE||||make new, transform, assay
|-
| pBca156-Bjh2344||2||2348||12 colonies||TO DO||V (UNSTABLE)||||make new, transform new and old, assay
|-
| pBca156-Bjh2344||2||2349||6 colonies||TO DO||V V||||make new, transform new and old, assay
|-
| pBca156-Bjh2344||2||2291||7 colonies||TO DO||V V||||make new, transform new and old, assay
|-
| pBca156-Bjh2344 with BAC||6||2331||tons of colonies||X||X||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh2344 with BAC||6||2333||tons of colonies||X||X||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh2344 with BAC||6||2348||1 colony (twice)||100-200 colonies||NOT DONE||||Today
|-
| pBca156-Bjh2344 with BAC||6||2349||1 colony(twice)||35-50 colonies||X||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh2344 with BAC||6||2291||35-50 colonies||100-200 colonies|| V V V||||transform new, assay
|-
| pBca156-Bjh1934||3||2331||35-50 colonies||X||X||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh1934||3||2333||<100 colonies||X||X||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh1934||3||2348||tons of colonies||X||V V||||transform old, assay
|-
| pBca156-Bjh1934||3||2349||tons of colonies ||X||V V||||transform old, assay
|-
| pBca156-Bjh1934||3||2291||tons of colonies||X||V ||||transform old,assay
|-
| pBca156-Bjh1934 with BAC||5||2331||tons of colonies||X||X||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh1934 with BAC||5||2333||tons of colonies||X||X||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh1934 with BAC||5||2348||tons of colones||X||X||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh1934 with BAC||5||2349||1o colonies||X||X||||\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
|-
| pBca156-Bjh1934 with BAC||5||2291||tons of colonies||X|| V V V||||transform old, assay
|}
*competent cell checks for new 1 and new 6 looked good.  
*miniprepped IBB as CA lefty, CK lefty and CK righty, and sent out for sequencing with ca998.


=='''To Do'''==  
=[[User:Tahoura Samad|Tahoura Samad]] 13:38, 1 July 2010 (EDT)=
*Remind Tim to streak more of the MG1655 '''DONE'''.
*controls for JTK030 looked good with no growth on Kan, Amp, Cam, good growth on LB, and a little growth on Spec, which is the normal result because of mutations in the bacteria. Those are labeled with double black stripes, in a box in the back of the -80 in the 2nd row from the top.
*Remember to pick the MG1655 colonies tomorrow. '''DONE'''
[[Image:IMG_5128.JPG|100px]] Kan<br>
*Pick and miniprep BAC from Germany '''DONE'''
[[Image:IMG_5129.JPG|100px]] LB <br>
*Pick and miniprep 0343, 0344, pBca1256-Bjh1934 '''DONE'''
[[Image:IMG_5130.JPG|100px]] Cam <br>
*Bring folder for iGEM assembly trees. '''DONE'''
[[Image:IMG_5131.JPG|100px]] Amp <br>
*coat hangers?
[[Image:IMG_5132.JPG|100px]] Spec <br>
*picked and grew up the results of PCA for the new nuclear localization tag. They had plenty of colonies, with only one background green colony. Will miniprep them tomorrow.
*did small scale competent cell prep of 1 and 6.
*did the following transformations (1old:2348), (5 old:2291) (6 old:2291) (6 new:2291),(3 old :2348) (3 old: 2349) (3 old:2291) (2 old:2348) (2 old: 2349)(2 old:2291) (2 new:2348) (2 new:2349) (2 new: 2291), (1 new:2331) (1 new:2333) (1 new:2348) (1 new:2349) (1 new: 2291) (6 new 2348) (6 new:2349), (IBB into lefty).
*redid assembly of stage 0 of life act but with 2.4 μL of each DNA and 1.2 μL of 5x NEB2.

Latest revision as of 14:10, 8 April 2011


Tahoura Samad 15:18, October 15 2010 (EDT)

-went down to Koshland to do confocal with Scott from the king lab. Could only load 30 ul of sample at time. Looked at NLS and 51 after induction fifteen minutes after feeding. -saw one possible hit, but set up wasn't optimal -choanos were suspended in low melt temp agarose. -probably better to use scope in Stanley. -lysis looks like it occured in pmll KC. miniprepped the plasmid, but probably will not use because it took so long. Also streaked out control, and will retest for lysis -for the future: JTK030 and 159 have different copy numbers. Probably why timing is different for 159 with 2801 and NLS which is 030. -New lifeact part 171 in 1600 still did not grow up

Tahoura Samad 15:18, October 14 2010 (EDT)

-started fresh choano culture by adding Kan to choano media culture. -picked colonies of AKS NLS with 51 and 52 as well as 2801 with 51 and 52 to look at under Koshland confocal. -checked on colonies of iGEM171 in 1600 (p15 spec). Did not grow up. Talk to Tim. -tested lysis of PCR of 51 digested and ligated in pmll KC. Did not appear to have lysed after 6 hours. kept in shaker

Tahoura Samad 15:18, October 13 2010 (EDT)

-set up test with NLS in 030 for 51 and 52 for confocal in upstairs stanley. -52 still behaving oddly, with little hits/fluoresence -got an excellent image of 51 at timepoint -15. showed fluoresence clearly filling a choano shaped object. YIPEEEEEE!!! -controls for new 11 cells looked clean. Transformations grew up alright. -controls for Daniela's 1600 payload cells looked clean.

Tahoura Samad 15:18, October 12 2010 (EDT)

-made competent cells of 11 and transformed them with 51 and 52 ran controls on AC, S, K.

Tahoura Samad 15:18, October 11 2010

-restransformed 51 . 52 into 9.

Tahoura Samad 15:18, 6 October 2010 (EDT)

-checked lysis Tecan with Daniela for iGEM171 with 51 and 52 in 1256. Appears to have lysed, just slowly. Tim is E/Bing it into p15a vector

Tahoura Samad 15:18, 5 October 2010 (EDT)

-fed choanos

Tahoura Samad 15:18, 4 October 2010 (EDT)

-did choano assays with 1934 NLS in JTK030 at induction time points +30, +15, 0, -15. -Had dones more hits in 51 for 0 and -15 time points. could barely move in field of view without seeing a hit! -52 did not have any hits at all.



=

Tahoura Samad 15:18, 24 September 2010 (EDT)

  • Started Eco/Bam of stuff into CK.
  • Mass production of choanos.

Tahoura Samad 18:36, Sept 23 2010 (EDT)

  • Ran assay with 51 and 52 in '8', which is Bjh1255 in JTK030, and '9' which is Bjh1934 in JTK030. Also tried to run Conor's 52 with iGEM159.
  • Didn't see invitro lysis for anything in 8, or with Conor's new lifeact construct.
  • Will transform over weekend so Tim can rerun assay on Wednesday.

To Do

  • Start mass production of choanos to run Josh's plasmids and Tera's plasmids.
  • Test lysis of Amy's CK stuff

Tahoura Samad 18:36, Sept 22 2010 (EDT)

  • Ran lysis of Amy's Stuff (51 and 52) which did not lyse after 2 hours
  • Tried to do lysis at room temperature. Induce at 11 am, and did not see lysis at 6 pm. Looks like we may have had lysis by the next day at around 10 am.
  • picked stuff for choano assays again tomorrow. 4 colonies each because of poor growth seen on Monday.

Tahoura Samad 18:36, Sept 21 2010 (EDT)

  • Choano care
  • ReKan'd a couple plates to get rid of biofilms.

Tahoura Samad 18:36, Sept 20 2010 (EDT)

  • Daniela accidently induced same sample twice, so scrapped '8' and '9' assay.
  • Ran choano assay on just 1968 in '11' with plain '11' as control. Didn't really see any of the same successful choano phenotype after 3 hours after feed at 90,60 and 30 min induction points. In addition, adding more bacteria (5ul vs 1ul) doesn't really appear to help efficiency. Just see a lot of stuff lysing outside of choanos. May not be limiting factor.

Tahoura Samad 18:36, Sept 19 2010 (EDT)

  • picked stuff for choano assay on Monday which includes 51 and 52 in 8 and 9, as well as Bjh1968, just self-lysis.

Tahoura Samad 18:36, Sept 18 2010 (EDT)

  • played with new iGEM friends with Christoph.
  • Out of five, only one, the Tetrahymena, appeared to have eaten the bacteria within the 4 hours we looked at them. At the final hour, samples were sluggish and swimming in erratic circles=not healthy.
  • Concerned about size being too big to see lysis even if it was occuring.

Tahoura Samad 18:36, Sept 17 2010 (EDT)

  • slept through self-lysis assay induction points. Will run on Sat or Monday.

Tahoura Samad 18:36, Sept 14 2010 (EDT)

  • ran same choano assay again. SUCESSS!!! Details in spreasheet. :D

Induction timepoints:

Tahoura Samad 18:36, Sept 10 2010 (EDT)

  • Look at choanos again after about 24 hours. FP signal is reallly low, takes about 15000 ms to really pick up anything in lysed samples. Not a lot of bacteria anywhere, especially in lysed samples.
  • Had Christoph transform again.

Tahoura Samad 18:36, Sept 9 2010 (EDT)

  • Ran choano assay. Induction starting at 10:15, with time points at 90,75,60,45,30,15. Fed at 12:00, So block was in shaker 15 minutes longer than expected. Started looking at approximately 3 pm.
  • GFP=300ms

Phase=162 ms

  • SUCCESS!!!!!! Saw green choanos in 51 at 90, 75 time points. Other samples were photobleached. Will save samples to look at again tomorrow and see if FPs last.


Tahoura Samad 18:36, Sept 8 2010 (EDT)

  • picked colonies to assay tomorrow. Grew up in TB. Will aspirate off ASW, and then resuspend in choano media.

Tahoura Samad 18:36, Sept 7 2010 (EDT)

  • Set up TECAN with LB, LB culture resuspended in ASW, Choano, 50:50, 25:75, 75:25 LB:ASW, and same for TB, TB resuspended.... S
  • Graphs show clear lysis for everything except for ASW and 50:50 for both 51 and 52.

Tahoura Samad 18:36, Sept 6 2010 (EDT)

  • Christoph ran lysis assays over weekend in LB/ASW and choano media. Said he saw lysis in all of them. Will run Tecan tomorrow to confirm.
  • Did microscopy assay of 51 and 52 induced 90, 75, 60,45,30,15 mins ahead of time after 24 hours. Did not see green phenotype anywhere, or really all that much green. Not a success, but not surprising considering that we don't really see lysis in ASW.

Tahoura Samad 18:36, Sept 3 2010 (EDT)

  • Microscopy comparing Tb vs LB 1, 2, 5, 10 ul.
  • Ran PCR of Pcons with Lifeact FP.
  • Gave payload project to conor.
  • Ran quick macroscale assay of lysis in ASW. Not apparent after four hours.

Tahoura Samad 18:36, Sept 2 2010 (EDT)

  • Had Christoph induce cells, started assaying at 3:00. Fatal error. Not enough bacteria for cells to eat. Will do a concentration microscopy test tomorrow to confirm.

Tahoura Samad 18:36, Sept 1 2010 (EDT)

  • sequencing results for 7 looked good.
  • picked colonies to feed and assay on Thurs.
  • ran lysis assay in LB for 51, and 52. Saw clear lysis after about 2 hours. 52 seems to lyse a little sooner then 51.
  • got two Pcon's from Tim/Sushant to put on the front of Lifeact. PCR off them with ca998 and g01001 and then Eco/Bam Eco/Bgl. Tape samples to the inside of TIm's fridge when done. WIll do tomorrow.

Tahoura Samad 18:36, 31 August 2010 (EDT)

  • miniprepped 3, 7, 8, and sent 7 in for sequencing with ca998.
  • controls for "11" cells looked good
  • picked cells to run preliminary lysis assay on Weds. before thursday

Tahoura Samad 18:36, 30 August 2010 (EDT)

  • Did colony 2K for Pbad. Lifeact GFP. Band should be around 2000. Will miniprep, 3, 7, 8 tomorrow.
  • Transformed 51, and 52 into "11" and JTK159 to replenish supply of PDD.
  • WIll assay on Wed, Thurs?

To Do

  • Miniprep 3, 7, 8 from col2k
  • pick colonies for assay on Wed. Either assay and invitro, or just invitro.
  • Sequence pbad. lifeactGFP.

Tahoura Samad 19:29, 27 August 2010 (EDT)

  • Made JTK2801 competent cells "11". Will run controls when I transform on Monday.
  • Make sure to email David Chen two days in advance about assaying.

Tahoura Samad 19:29, 26 August 2010 (EDT)

  • Analyzed Tecan data Tim. 51 and 52, along with 41 lysed. Will hopefully will be continuing lysis assays with 51, 52.
  • Eco/Bam Eco/Bgl with Neb3 AraC. Pbad with Lifeact RFP.
  • picked up JTK8021 to make comp cells with and assay.
  • picked Bjh2252 to miniprep and assemble with AraC.Pbad.

To Do

  • ReEcoBam 2271 into CA. First check with Tim though.
  • Check with Tim about assaying when he's gone
  • Check with Tim what the heck to do with AraC. Pbad LIfeact
  • Make comp cells with JTK8021.

Tahoura Samad 19:29, 25 August 2010 (EDT)

  • Ran lysis assay with 41, 42, 45-52 in JTK030, and in 159 3 different colonies. Will analyze Tecan Data tonight and tomorrow.
  • Talked to Tim about Lifeact parts. Lifeact GFP sequenced well but is not green. Abandon for now. LifeactRFP is heavily mutated in the Lifeact Region. Abandon
  • Will start assembly of Pbad. with LIfeact GFP and RFP. Grow up titration curve? Talk to Tim

Tahoura Samad 19:29, 23 August 2010 (EDT)

  • 2ab assembly products were not green and red. Will ask Daniela to pick on Tuesday to miniprep and see what is going on.
  • Neb2 Eco/Bgl LifeactRFP are now appearing red. Tim miniprepped them for me. Sent them in for sequencing to see what is going on.
  • Talked to Daniela and Tim about end of lysis assay on Friday. (everything in JTK159, plated on both Kan and Amp. Looks like just 51 and 52 lysed.
  • Retransformed 41, 42, 45-52 into Jtk159 and Jtk030. Prepped blocks for Daniela to pick tomorrow so I can assay on Weds.
  • Picked BJh2541, Bjh2252 Eco/Bgl in Neb 2 and Bjh2251 by 2ab for Daniela to miniprep tomorrow.
  • Also transformed Neb3 EcoBgl assembly products.

Tahoura Samad 19:29, 20 August 2010 (EDT)

  • Ran lysis assay in TB for 41, 42, 45-52. Left early to move in and will get results from Daniela.

Tahoura Samad 16:35, 18 August 2010 (EDT)

New Payload Work

  • Saw growth on plates, but no green and red colonies, so assembly was a failure.
  • Redid shortcut method of assembly, but digested EcoBgl with NEB3 instead of NEB 2.
  • Also created new assembly trees and Eco Bammed everything into pmll vectors.
  • Retransformed 41, 42, 45-52 and plated on AC for 41 and 42 and AK for 45-52 to avoid contamination issues. Asked Daniella to pick them for me tomorrow. Daniela said she would pick colonies for EcoBam stuff and do 2ab assembly on Friday. I'll do lysis testing.

Tahoura Samad 16:35, 17 August 2010 (EDT)

New Payload Work

  • transformed everything from yesterday

Biochipper's Device

  • tried and failed at loading it. Maybe tape seal was no good?

Lysis

  • redid Amy's lysis assay in TB with no sucess. Amp plates seem to be contaminated and we plated on Amp. only. Will retransform 41, 42, 45-52 tomorrow.

Tahoura Samad 16:35, 16 August 2010 (EDT)

New Payload Work

  • retried Christoph and Conor's shortcut for assembly of the two new payloads by PCRing of PCON from Josh (plate 5, well c10) with phusion. Expected band length was 1000 bp. Cut and gel purified, and digested with Eco Bam. Also digested Lifeact GFP and RFP from Jin with Eco Bgl. Ligated Pcon eco bam into Eco/bgled lifeact rfp and gfp and transformed into Pir Lefty.

Lysis Testing

  • Amy ran lysis test and saw no lysis. Will rerun test for her in just TB tomorrow.

Tahoura Samad 16:35, 13 August 2010 (EDT

  • split choanos
  • made JTK159 cells.

Tahoura Samad 16:35, 12 August 2010 (EDT)

  • Met up with Gayla and Tigran to learn how to make chips with Daniela. Practiced pouring PDMS, cutting devices, bonding etc. Make sure to get Gayla's guide.
  • Found out that our jtk030 cells also have ptet in front of pir, which is leading to other problems. Tim grew up some JTK159, and we will make cells tomorrow.

Tahoura Samad 16:35, 11 August 2010 (EDT)

  • found out that all our payloads have pTet, which means they are useless with TetR. Need to make two new payloads. Abandoned assembly with new iBB because it includes pTet in front of GFP.
  • Helped Amy wash samples for self-lysis test without payload.
  • Ran TECAN along with macro scale test. Saw lysis for several constructs! See excel sheet.
  • Also did stickiness assay to confirm lysis.

Tahoura Samad 16:35, 10 August 2010 (EDT)

Tahoura Samad 9:25, 9 August 2010 (EDT)

  • set up lysis assay. saw no lysis after 3 hours. grew upstairs in shaker.
  • Choanos do not like to be left over the weekend.
  • picked stuff for assembly final step.

Tahoura Samad 18:32, 8 August 2010 (EDT)

  • picked colonies for lysis assay on Monday and grew up in TB
  • tested 1934 comp cells by transforming with 47.

Tahoura Samad 11:25, 6 August 2010 (EDT)

Choano Assay

  • Set up lysis assay for 47 and 48 with payload in LB vs in ASW, with Mc1061 with and without ATC as a control. Results were that both control and positive ATC OD dropped off at the same rate, possibly because the temperature control on the TECAN is at 26 degrees rather than 37.
  • Lifeact RFP didn't grow up very well so was unable to get it to grow up well enough to make comp cells. Will retransform next week.

Tahoura Samad 11:25, 5 August 2010 (EDT)

Choano Assay

  • growth of cells in LB was poor, which confirms that life-time on each transformation is less than a week. Ideally within the first four days of life of the plate. Set up chonao assay again but saw no lysis. Will redo in TECAN tomorrow using TB to get better growth.
  • made competent cells for Lifeact GFP in JTK030 and Bjh1934 in JTK030.

Choanos

  • none of choano cultures, including 1:2 was at assaying density yet. Fed each 40 ul and let sit.

Tahoura Samad 9:32, 4 August 2010 (EDT)

Choano Assay

  • Set up chonao assay. Washed saturated LB culture of 47 and 48 with payload delivery in choano media, ASW, TB and LB. Using 2 3 mL aliquots, added ATC to one tube and none to other to serve as control. DId not see lysis after 3 hours. Will grow up in LB and redo test on Thursday.
  • re-cotransformed JTK030 with Bjh1934 and pBca1256 for assaying this week. Transformed JTK030 with lifeact GFP and RFP and 1934to make comp cells.

IBB

  • Eco Bammed RFP Boo15 out of hybrid plasmid into KC, and transformed into MC1061 pir righty.
  • Quintara resequenced Rbs ibb, gfp and it looks perfect. Tim deleted the first three bp off the oligo, so that's why there is that deletion in the front of the rbs.

Choanos=

  • obtained a saturated choano culture. Split 1:2, 1:5, !;15 and fed 40 ul of choano smoothie. Will observe growth as compared to choanos in choano media.

Tahoura Samad 13:32, 3 August 2010 (EDT)

Choano Assay

  • ran assay of taking pictures of choanos eating just payload bacteria every five minutes for an hour. Saw photobleaching at a round minute 82. Arrived at scope 15 minutes after having fed choanos and were not seeing any rod phenotype. Hard to conclude the details of choano digestion because choanos keep moving slightly in and out of plain, messing up focus of FPs. Need to figure out how to fix choanos in space.
  • miniprepped Rfp terminator and GFP rbs ibb. RFP terminator mapped strangely. Its a hybrid plasmid. Solution: PCR off hybrid plamsid using Ca998, G0100, digest, and ligate into correct plasmid.

Lysis Assay

  • Did a loose assay to check if construct 47 and 48 without payload were lysing in ASW. Grew up in LB to saturation, washed and added ATC. Did not lyse. Picked and grew up colonies in TB to redo assay tomorrow. Will use just TB culture as a control, because it should lyse obviously within two hours according to Amy.

=

Tahoura Samad 16:03, 2 August 2010 (EDT)

Chonao Assay

  • Checked 3 day culture of flavobacterium free chonaos. Not as dense as a 3 day culture should be.
  • Ran assay of TetR 47, TetR48 at +15, 0 -15 and -30. After 1 hour, seeing some rod shaped bacteria, but some empty choanos. Didn't see desired phenotype after 1 or 4 hours. Images are clearer without biofilms though.
  • Surprised not to see some of landmarks of digestion we expected to see. Rounding up? Run 1 hour assay tomorrow to establish baseline

Other

  • streaked out JTK030. Pick to make comp. cells tomorrow. Make iGEM glycerol stock of Jtk030.

Lifeact

  • picked LIfeact GFP to make comp cells today. In JTK030.
  • picked LIfeact RFP in 1256

Tahoura Samad 16:03, 29 July 2010 (EDT)

Choano Assay

  • began set up for choano assay at 10:00.
  • used whole mLs for each of three hour time points because of loss of choanos during washing.
  1. 10:15 am, made 3 1 ml aliquots of each TetR 47 and TetR 48. TetR 47 did not grow up as well as TetR 48.
  2. 10:30 added ATC to samples with ATC for 90 minutes. (1 μL per mL of bacteria, keep in shaker.)
  3. 10:45 split choanos 1:15.
  4. 11:00 Added ATC to samples with ATC for 60 minutes. Started filtering choanos
  5. 11:30 Added ATC to samples with ATC for 30 minutes.
  6. 11:40 Added NH4CL to samples that needed it.
  7. 12:00 Fed choanos. Took about 10 minutes.
  8. 12:35 Washed choanos with 1 mL of choano media and chased with 25 mL of white ecoli (plain MG1655)
  9. 1:00 Started filtering choanos
  • looked at chonaos at around the two hour mark. Very little fluorescence at all, only getting fluoresence when we bumped up the exposure to like 500 ms. Possible that the cells are lysing way before they are eaten. Chris says to try intervals between 15 minutes before to 15 minutes after feeding.
  • in assay, chase with less white ecoli.
  • from assay, can conclude that choanos complete digestion definitely within 2 hours, probably within 1.
  • no such thing as narchoanos. :( just choanos with two flagella.

IBB Work

  • miniprepped 3 of 4 colonies that weren't cotransformed for assembly without ATP and with DNTPs.
  • picked and grew up Bca1092

To Do

  • streak out JTK030 from somewhere
  • transform 1934 into JTK030 and make into competent cells.
  • transform 2255 into JTK030 and make into competent cells.
  • EcoBam 2254 again and transform into JTK030.
  • redo assembly of GFP RBS ibb.
  • miniprep BCA1092 and do assembly of RFP terminator
  • pick and grow up 47 to miniprep, 48
  • miniprep 1934 payload.

Tahoura Samad 15:12, 28 July 2010 (EDT)

Tahoura Samad 15:12, 27 July 2010 (EDT)

IBB Work

  • miniprepped RFP KA and mapped it to make sure it wasn't methylated at bgl and bam.

eco bam of rfp bam xho bgl xho. looks good

  • got BCA1092 from Jin and transformed it into righty.
  • miniprepped TIm's GFP.
  • started assembly of AC Tim's GFP with CK rbs ibb.
  • sent RBS ibb in for sequencing.

Lifeact

  • Lifeact RFP transformations came up green, which means i used the wrong DNA. Redid Eco Bam and transformed into MG1655.

Choano Assay

  • had Conor plate my transformations, but I'm worried about them actually being co transformed because top plated spec and you can see that they are all growing up around the edges, where there might not be spec. Fishy. Redid cotransformations with 1 ul and 3 ul of straight DNA of each.

Tahoura Samad 13:11, 26 July 2010 (EDT)

Choano Assay

  • TetR 47 and 48 didn't grow up because I transformed them into cells without pir. They are not in PMLL, but in entry vectors. Redid those transformations today. (cotransformation)
  • filtered chonaos, fed with 1 μL of overnight culture, waited 20 minutes, washed and then chased with 15 μL of white bacteria.
  • observed after 1 hour, 4 hours. Still too much green bacteria outside cells, looks like they are still eating. Lots of rod-shaped. Strangely, not a lot of degradation of gfp after 1hour in cells without nh4.
  • lifeact: after 1 hour, cells are green, can see rod shaped bacteria. After four hours, choanos look pretty empty (digested?) Interesting to note that several times we observed choanos with extremely round dot of green adjacent to them? Also saw balls of green in their bodies. Actin is balling up?

IBB

  • did Eco/Bam of Lifeact RFP into 1256 (f4 on TIm's stock plate 1) background will be white. Will transform tomorrow.
  • picked and


Tahoura Samad 13:11, 22 July 2010 (EDT)

PDD Subgroup Work

  • test for competency of 3s was interesting. Cells not as good as original with 25 colonies for 2349 and about 50 for 2333. Might remake cells. Probably let them sit out for too long.

IBB

  • digested rbs. ibb and gel purified product. (small fragment)

Lifeact

  • transformations worked. Will pick 2255 on Sunday, make competent cells on Monday.

To Do

  • Pick 3s, Bjh2255 on Sunday.

Tahoura Samad 12:50, 21 July 2010 (EDT)

  • Lifeact assembly failed again. Found out that Jin has GFP and RFP versions of the part I'm trying to assemble. Abandoned LIfeact Assembly.

bgl/xho
bam xho, eco, xho

  • Got Lifeact RFP and Lifeact GFP from Jin. Transformed Lifeact RFP into MC1061 and Lifeact GFP (Bjh2255)into MG1655. Lifeact RFP 2254 needs to be ecobam transfered into pbca1256.
  • gel purify PCR product, digest and ligate into vector, retransform. Digestion done at 11:06 pm. Gel came up empty, so redid PCR wit 1:10 dilution of olios.
  • do test transformation with 3s to make sure they work equally well as the last ones with 2333 and 2349.
  • transformed Amy's new constructs into 3s. Set up choano assay for Monday.
  • Get parts from Jin
  • Figure out 1934 payload restructure.
  • controls for competent cells looked good, with no growth on Cam, Kan and Amp and growth on LB and Spec.

LB
Cam
Amp
Kan
Spec


To Do

  • transform some of JIn's stuff into cells. Pick, grow up, miniprep. Eco Bam 2254them into 1256.
  • re _eco bam transfer RFP!
  • digest, ligate etc rbs.ibb and send for sequencing.

Tahoura Samad 17:03, 20 July 2010 (EDT)

  • Lifeact failed to assemble.
  • made competent 3 cells
  • talked to Chris about choanos test with just GFP.
  • Redid Lifeact assembly.

Tahoura Samad 17:03, 19 July 2010 (EDT)

  • Assembly of LIfeact part
  • set up FACs training.

Tahoura Samad 17:03, 16 July 2010 (EDT)

PDD Work

  • did transformations of 3s with PDD
  • did transformations of MG1655 with Tim's RFP (a5, plate 2). Could plate that after heatshocking because its amp.
  • streaked out 3s and 5s

To Do

  • Saturday: pick and grow up 3s,Mg1655 with RFP.
  • Sunday: assay. Split choanos 1:5 so that we have some for monday.
  • Sunday:pick and grow up RFPs so that we have samples to look at under flow cytometer.
  • Monday: split choanos 1:15

Tahoura Samad 16:46, 15 July 2010 (EDT)

PDD Subgroup Work

  • assembly of LifeAct abandoned. Now up to Christoph and Daniela
  • learned how to use microscope at 10 am (Arkin Lab). Looked at choanos after 1 hour and got pictures
  • set up time scale for weekend.

IBB

  • miniprepped IBB in CK Righty and CA Righty. Will not sequence.
  • go over assembly tree of that with TIm.
  • got rbs oligo and resuspended at 100 mM (nm times 10 μL.)

To Do

  • transformations of 3s with PDDs
  • transformation of RFP into Mg1655
  • start PCR of rbs onto IBB.
  • split choanos so i have 50 mls on Sunday.
  • streak out 3s and 5s

Tahoura Samad 13:48, 14 July 2010 (EDT)

PDD Subgroup Work

  • picked and grew up 3 series for microscopy
  • picked and grew up 3s and 5s for minimal media test. Same procedure as last time, but no duplicates, and grow up in minimal media with 10 mM MgSo4 overnight for 16 hours.

Additional

  • Checked controls for Pir1 MC1061 lefty and rightys. Controls looked good.

IBB Work

  • Miniprepped Ptet and b1006 for assembly trees
  • Checked with TIm how to make oligo to put rbs on the front of ibb and made oligo. Temo of annealing region should be 55 c. THe nonsense sequence in front of the rbs is just random stuff Tim put there.

Lifeact

  • miniprepped lifeact that is actually KA and mapped to make sure it was correct. Expected sizes 1300, 1700

ca ka ck

.

To Do

  • Jin wants to redo the minimal media test, but not in duplicate and make two aliquots after filtering the choanos.
  • Pick 3 and 5 on Tuesday
  • Split chonaos 1:15 on thursday again
  • Pick and grow up CK Righty, CA Righty,RFP! and 3s.


Tahoura Samad 13:56, 13 July 2010 (EDT)

PDD Subgroup Work

  • started Eco Bam transfer of 1882 part for lifeact tree into AK.
  • sent Ptet+Lifeact in for sequencing. We suspect it is bad because one of the inputs was in KA, which was actually AK.
  • Talked over results of minimal media testing with Jin. Results were ambiguous because there was inconstancies between the duplicates. Additionally, Jin thinks that Mg in the choano media may be obscuring the results. One thing he observed that he wants to investigate further is that he thinks 37 C might be slowing down the choano's digestion.
  • Split chonaos 1:2 so they will be ready for assaying on Thursday
  • Started transformations of 3s for practice assay on Thursday Morning. Talked to Jin. He said transformations are good for about 3 days on plate.
  • transformed CA IBB eco bam transfer into Righty cells and CK IBB DNA (old) into righty cells.

Additional

  • Checked controls for Pir1 MC1061 lefty and rightys. Controls looked good.

Kan
Spec
Cam
Amp
LB

IBB Work

  • transformed CA IBB eco bam transfer into Righty cells and CK IBB DNA (old) into righty cells.
  • Made assembly trees of parts with Tim.
  • picked some of Ptet and bl006 of Christoph's plates for my assembly.

To Do

  • Jin wants to redo the minimal media test, but not in duplicate and make two aliquots after filtering the choanos.
  • Pick 3 and 5 on Tuesday
  • Split chonaos 1:15 on thursday again
  • Pick and grow up CK Righty, CA Righty,RFP! and 3s.


Tahoura Samad 17:20, 12 July 2010 (EDT)

  • ran 1B of colony pcr for life act again. looked good. will send for sequencing tomorrow
  • ran Jin's minimal media assay
  • Eco Bam transfered Ck lefty into Ca. will transform into righty cells.

Tahoura Samad 17:20, 9 July 2010 (EDT)

  • miniprepped col PCR results, which grew up equally poorly. May have a hit on the first one. Will transform on Monday

  • did transformations of 3s with 5 pdds.
  • set up wells so amy can pick on Saturday

To Do

  • Sunday: split choanos 1:5 so i have enough for Monday's testing. Set up assay for Jin, filter after 2hours. Pick and grow up 3s and 5s for 16 hrs for minimal media testing
  • pick Pir1 lefty and righty for competent cell prep.

Tahoura Samad 15:18, 8 July 2010 (EDT)

PDD Subgroup Work

  • direct Jin to confirm which of the combinations is actually working.
  • remind Tim to email Jin about scope.

Life Act

  • Even after 24 hours, the bacteria from the colony PCR still did not grow up well. Only saw growth for a couple. Miniprepped colonies 6, 9, 19, 20. Restriction mapped Bam, Eco, excpected band length of 167 and 2700

Other

  • picked MC1061 pir1 lefty and righty and am growing up in LB.

Tahoura Samad 16:10, 7 July 2010 (EDT)

PDD Subgroup Work

  • nothing

LifeAct

  • redid colony PCR with 24 new colonies. Results looked good, with many bands at expected length of 357.

  • will miniprep in the afternoon.

Other

  • streaked out MC1061 pir lefty and righty since we're almost out. Will make competent cells on Friday.

To Do

  • Pick and grow up Pir Lefty and Righty for competent cell prep on Friday.
  • On Friday, do transformations of all 3s with all five PDDs.
  • On Friday, make competent cells.
  • On Saturday, take out of incubator, pick and grow up.
  • On Sunday, feed to choanos.
  • On Sunday, pick and grow up 3 and 5 for Monday.
  • ask Tim what to do with IBB.
  • get solutions of MMM and MM0 from Jin.
  • ask Jin #grow up in shaker for 2 more hours. (Ask Jin what how much of each solution to grow them up in.)
  • split choanos 1:5 on Sunday, so we'll have enough for Monday. Check with conor how to get 17 mL of choanos for Monday.

Tahoura Samad 18:16, 6 July 2010 (EDT)

PDD Subgroup Work/Discussion With Jin

  • talked to Jin. He was not able to look at the combinations from Friday. :(
  • Jin says to let all the different combinations rest, and focus on the 1934 payload.
  • Jin also says to start the minimal media testing to see if pb6 works.

Minimal Media Testing

  • Day 1
  1. Streak out 3, 5
  2. Pick and grow up in TB with 30 mM MgS04 (30 μL per mL) overnight.(16 hrs) Do two colonies of each 3 and 5.
  • Day 2
  1. Dilute 100 fold by adding 30 μL of overnight culture to 3 mL of TB. (4 tubes, 2 of 3 and two of 5)
  2. Grow for 2-3 hours more. (2 hours)
  3. make 2. 1 mL aliquots of 3 and 5. (Four of each) (8 tubes total)
  4. Wash 3 times.
    1. Wash two of each with minimal media with magnesium (MMM) and Minimal media without magnesium. (MM0) Get these solutions from Jin.
    2. When you wash these guys, spin the cells down, carefully pipette off the supernatant without touching the pellet, resuspend with solution etc .3 times.
  5. grow up in shaker for 2 more hours. (Ask Jin what how much of each solution to grow them up in.)
  6. filter 17 mL of choanos
  7. incubate 8 mL of choanos with 10 μL per mL (10 mM ) NH4Cl for 10 minutes.
  8. feed 10 μL of bacteria to choanos. Look at after 5 hours.


Lifeact

  • ran colony PCR for 32 colonies.

  • minipreped 30 and 32, but they turned out to be contransformed. Of 32 colonies, 7 were contransformed.

NLS

  • sequencing results looked okay.
  • iGEM_028, CK lefty looks perfect except for a point mutation in the EcoR1 site. Looking at the abi, i think it might be a miscall.
  • iGEM_029, CK Righty looks perfect except for a one base deletion after the Pts1 site. Looking at the Abi file, exactly at this point, the sample gets mixed reads. ?
  • iGEM_030, CA Righty looks perfect except for the point mutation in the Ecor1 site. Looking at the abi, i think it might be a miscall.

To Do

  • On Friday, do transformations of all 3s with all five PDDs.
  • On Saturday, take out of incubator, pick and grow up.
  • On Sunday, feed to choanos.
  • On Sunday, pick and grow up 3 and 5 for Monday.


Tahoura Samad 13:22, 5 July 2010 (EDT)

  • picked and grew up 1, 2, 6, 7, to make competent cells.

Tahoura Samad 13:22, 2 July 2010 (EDT)

  • most of my transformations with the new competent cells were successful.
Competent cell Number PDD Growth in initial competent cells Growth in Small Scale Competent Cells Result of Assay with Jin Conclusion
pBca156-Bjh2343 1 2331 0 tons of colonies NOT DONE transform new, assay
pBca156-Bjh2343 1 2333 0 tons of colonies NOT DONE transform new, assay
pBca156-Bjh2343 1 2348 1 colony (twice) tons of colonies NOT DONE transform new, assay
pBca156-Bjh2343 1 2349 0 50-100 colonies NOT DONE transform new, assay
pBca156-Bjh2343 1 2291 0 100-200 colonies NOT DONE transform new, assay
pBca156-Bjh2343 with BAC 7 2331 0 15 colonies NOT DONE Today
pBca156-Bjh2343 with BAC 7 2333 0 2 colonies NOT DONE Today
pBca156-Bjh2343 with BAC 7 2348 0 4 colonies NOT DONE Today
pBca156-Bjh2343 with BAC 7 2349 0 0 DEAD \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2343 with BAC 7 2291 0 0 DEAD \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344 2 2331 0 tons of colonies NOT DONE make new, transform, assay
pBca156-Bjh2344 2 2333 0 tons of colonies NOT DONE make new, transform, assay
pBca156-Bjh2344 2 2348 12 colonies TO DO V (UNSTABLE) make new, transform new and old, assay
pBca156-Bjh2344 2 2349 6 colonies TO DO V V make new, transform new and old, assay
pBca156-Bjh2344 2 2291 7 colonies TO DO V V make new, transform new and old, assay
pBca156-Bjh2344 with BAC 6 2331 tons of colonies X X \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344 with BAC 6 2333 tons of colonies X X \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344 with BAC 6 2348 1 colony (twice) 100-200 colonies NOT DONE Today
pBca156-Bjh2344 with BAC 6 2349 1 colony(twice) 35-50 colonies X \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344 with BAC 6 2291 35-50 colonies 100-200 colonies V V V transform new, assay
pBca156-Bjh1934 3 2331 35-50 colonies X X \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 3 2333 <100 colonies X X \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 3 2348 tons of colonies X V V transform old, assay
pBca156-Bjh1934 3 2349 tons of colonies X V V transform old, assay
pBca156-Bjh1934 3 2291 tons of colonies X V transform old,assay
pBca156-Bjh1934 with BAC 5 2331 tons of colonies X X \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC 5 2333 tons of colonies X X \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC 5 2348 tons of colones X X \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC 5 2349 1o colonies X X \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC 5 2291 tons of colonies X V V V transform old, assay
  • competent cell checks for new 1 and new 6 looked good.
  • miniprepped IBB as CA lefty, CK lefty and CK righty, and sent out for sequencing with ca998.

Tahoura Samad 13:38, 1 July 2010 (EDT)

  • controls for JTK030 looked good with no growth on Kan, Amp, Cam, good growth on LB, and a little growth on Spec, which is the normal result because of mutations in the bacteria. Those are labeled with double black stripes, in a box in the back of the -80 in the 2nd row from the top.

Kan
LB
Cam
Amp
Spec

  • picked and grew up the results of PCA for the new nuclear localization tag. They had plenty of colonies, with only one background green colony. Will miniprep them tomorrow.
  • did small scale competent cell prep of 1 and 6.
  • did the following transformations (1old:2348), (5 old:2291) (6 old:2291) (6 new:2291),(3 old :2348) (3 old: 2349) (3 old:2291) (2 old:2348) (2 old: 2349)(2 old:2291) (2 new:2348) (2 new:2349) (2 new: 2291), (1 new:2331) (1 new:2333) (1 new:2348) (1 new:2349) (1 new: 2291) (6 new 2348) (6 new:2349), (IBB into lefty).
  • redid assembly of stage 0 of life act but with 2.4 μL of each DNA and 1.2 μL of 5x NEB2.
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