Berk2010-Tahoura

|Robot June

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, 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.

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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.