Berk2010-Tahoura

From OpenWetWare

Revision as of 13:48, 10 August 2010 by Tahoura Samad (Talk | contribs)
Jump to: navigation, search

June


Contents

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)

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.

=

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

Image:IMG_5355.JPGca 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-Bjh2343123310tons of coloniesNOT DONEtransform new, assay
pBca156-Bjh2343123330tons of coloniesNOT DONEtransform new, assay
pBca156-Bjh2343123481 colony (twice)tons of coloniesNOT DONEtransform new, assay
pBca156-Bjh234312349050-100 coloniesNOT DONEtransform new, assay
pBca156-Bjh2343122910100-200 coloniesNOT DONEtransform new, assay
pBca156-Bjh2343 with BAC72331015 coloniesNOT DONEToday
pBca156-Bjh2343 with BAC7233302 coloniesNOT DONEToday
pBca156-Bjh2343 with BAC7234804 coloniesNOT DONEToday
pBca156-Bjh2343 with BAC7234900DEAD\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2343 with BAC7229100DEAD\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344223310tons of coloniesNOT DONEmake new, transform, assay
pBca156-Bjh2344223330tons of coloniesNOT DONEmake new, transform, assay
pBca156-Bjh23442234812 coloniesTO DOV (UNSTABLE)make new, transform new and old, assay
pBca156-Bjh2344223496 coloniesTO DOV Vmake new, transform new and old, assay
pBca156-Bjh2344222917 coloniesTO DOV Vmake new, transform new and old, assay
pBca156-Bjh2344 with BAC62331tons of coloniesXX\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344 with BAC62333tons of coloniesXX\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344 with BAC623481 colony (twice)100-200 coloniesNOT DONEToday
pBca156-Bjh2344 with BAC623491 colony(twice)35-50 coloniesX\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344 with BAC6229135-50 colonies100-200 colonies V V Vtransform new, assay
pBca156-Bjh19343233135-50 coloniesXX\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh193432333<100 coloniesXX\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh193432348tons of coloniesXV Vtransform old, assay
pBca156-Bjh193432349tons of colonies XV Vtransform old, assay
pBca156-Bjh193432291tons of coloniesXV transform old,assay
pBca156-Bjh1934 with BAC52331tons of coloniesXX\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC52333tons of coloniesXX\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC52348tons of colonesXX\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC523491o coloniesXX\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC52291tons of coloniesX V V Vtransform 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.
Personal tools