June

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Tahoura Samad 13:57, 30 June 2010 (EDT)

PDD Subgroup Work

  • did large scale competent cell prep of JTK030 again.
  • did small scale competent prep of 1, 2, 6, 7. Inoculated 40 μL into 4 mL of LB and grew up for three hours. 7 and 2 grew up well and had large pellets. 1 had a tiny pellet, and was chucked. 6 had a moderate sized pellet, but i transformed anyway. Combinations I transformed and plated were 7 with 2331, 2333, 2348, 2349, 2291, 6 with 2348 and 2349. 2 with 2331, 2333. Rescued with TB. After incubation, only the two 6s looked unhealthy, with relatively clear liquid.
  • digested PCA product, ran a gel. Band should have been at about 250. It was, but was smeary and wide. Cut it out anyway, gel purified, ligated into CK, CA (pmll5, pmll9, which are predigested).
  • transformed CK into lefty and righty, and CA into just righty.
  • repicked and grew up red colonies from 6 and red colonies from 1 to do small scale competent cell prep.
  • Did assembly of lifeact with Christoph, using 1.2 μL of DNA and 5 μL of 5x NEB2 buffer. Transformed it into lefty cells and plated.

Tahoura Samad 13:57, 29 June 2010 (EDT)

PDD Subgroup Work

  • checked streaked out plates. 1 had poor growth, with three colonies.2 had good growth, 6,7 had moderate growth with mixed phenotypes.
  • picked two colonies from each plate to grow up and do small scale competent cell prep.
  • some of the combinations worked vacuole busted! :D
  • Started PCA of new nuclear localization tag. This one attaches to protein B of the protein complex, versus protein A. Conor designed the oligos for these. (iGEM oligos 37-44).
  • started assembly of lifeact part:{Ptet}{rbs_LifeAct_GDPPVAT>}{<RFP!}. According to Tim, lifeact is a tag that associates with Actin, which is part of the

eukaryotic cytoskeleton. We want to see if we can light that up with RFP to make sure the choanos are still alive and as a check that delivery is actually occurring. Check with Cristoph where to find these parts, what vectors (A,K...etc) they are in, as well as for the protocol.

  • stopped PCA after zymoing the second amplification with outer primers.

Other

  • checked controls for JTK030.
  • Controls were bad, with growth where there shouldn't have been on Spec and Amp. Josh says he things he grew up cells in Spec contaminated LB.

Negative control for Amp had growth
Negative control for Spec had growth
Positive Control with plain LB
Negative Control for Kan
Negative Control for Cam

  • picked JTK030.

To Do

  • Large Scale competent prep of JTK030
  • Small Scale competent prep of 1, 2, 6, 7 and transformation. Make 2 ;4 mL preps for each.
  • Finish PCA (digest, cut, gel purify transform etc.
  • Ask Jin which specific combinations worked.
  • talk to Christoph and start assembly of LifeAct with RFP.

Tahoura Samad 14:48, 28 June 2010 (EDT)

PDD Subgroup Work

Family # No Growth Growth No Growth Growth
Bjh2343123312348: 1 colony22912291: 7 colonies
233322912291: tons of colonies
234923312291:35-50 colones
229123312291:tons of colonies
7233123312331:35-50 colonies
233323332331:tons of colonies
234823332331:tons of colonies
234923332333: >100 colonies
229123482333: tons of colonies
Bj2344223312348:12 colonies23492333:tons of colonies
23332349: 6 colonies23492348: 1 colony
2291: 7 colonies2348: 1 colony
62331:tons of colonies2348: tons of colonies
2333:tons of colonies2348: tons of colonies
2348: 1 colony2348:12 colonies
2349: 1 colony (twice)2349: 1 colony (twice)
2291:35-50 colones2349: 10 colonies
Bjh193432331:35-50 colonies2349: 6 colonies
2333: >100 colonies2349: tons of colonies
2348: tons of colonies
2349: tons of colonies
2291: tons of colonies
52331:tons of colonies
2333: tons of colonies
2348: tons of colonies
2349: 10 colonies
2291:tons of colonies
  • based on observations, minipreps are fine.
  • maybe certain combinations are toxic.
  • streaked out 1,2,6,7.

Choano Assays

  • Couldn't look at these with Jin, check what happened
  • Combinations Jin looked at are 1 with 2348, 2 with 2348,2349,2291, 2 with 2331,2333,2348,2349,2291, 5 with 2291, 6 with 2291. Jin says some of them worked! (green cytoplasm, red nucleus means it busted out of the vacuole. He thinks the nuclear localization might not be so good though.

To do

  • take cells to assay out at 2 pm and feed to choanos.
  • start competent prep of Jtk030 at 2:35 pm. Set up controls on LB, Spec, Cam, Kan, Amp.
  • small scale competent prep of 1, 2, 6,7.


Tahoura Samad 12:55, 25 June 2010 (EDT)

PDD Subgroup Work

  • 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.
  • miniprepped pBca1601-Bjh2291.
  • Started transformations. Combinations include 2331:1) , (2333:1), (2348:1), (2349:1), (2291:1), (2331:2, 2333:2), (2348:2), (2349:2), (2291:2), (2331:3), (2333:3), (2348:3), (2349:3), (2291:3), [15 combinations, 9 plates] and (2291:5), (2291:6), (2291:7), and (2331:7) . It is interesting to note that after rescuing, all the ones and twos and 7s came out very clear, while the 3s, 5s and 6s cloudy.

To Do

  • Shelley is taking plates out on Sat. room temp.
  • (2331:1) , (2333:1), (2348:1), (2349:1), (2291:1), (2331:2, 2333:2), (2348:2), (2349:2), (2291:2), (2331:3), (2333:3), (2348:3), (2349:3), (2291:3), [15 combinations, 9 plates] need to be grown up in Amp,Kan,Spec TB and magnesium.
  • (2291:5), (2291:6), (2291:7), and (2331:7) [4 combinations, 2 plates] need to be grown up in Amp, Kan, Spec, Cam TB and magnesium. There should be a falcon tube of A,K,S,C TB and magnesium in the drawers, but i haven't poured out a falcon tube of A,K,S TB yet. The TB bottle is up on the shelves above the bench.

Tahoura Samad 14:15, 24 June 2010 (EDT)

PDD Subgroup Work

  • picked AK transformations of Bjh2291.
  • 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?
  • 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.

To Do

  • miniprep 2291
  • 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.
  • Ask

Tahoura Samad 19:20, 23 June 2010 (EDT)

PDD Subgroup Work

  • 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.
  • Also, transformed and plated E/B transfer of 2291. Checked for background, flourescent green cells, and marked them.

Choano Assays

  • started choano microscopy 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.
  • Jin looked at 5 with 2331,2333,2348,2349 and 6 with 2331, 2333 with and without NH4Cl. Jin says that the Mg is preventing the pb6 from working as well as it should. Additionally, the Nh4Cl is slowing down the choanos. He thinks this because the GFP appears to degrade more quickly then RFP (degrades at a higher pH. In the non NH4Cl cells, after 2 hours of incubation, there was almost no GFP and a little bit of RFP in small circular vacuoles. In the cells with NH4Cl, there were almost equal amounts of GFP and RFP, which would indicate that the cell metabolism is being slowed down.

To Do

  • pick colonies up if they grow. Grow up in TB and magnesium at the end of the day.
  • test cam/spec plates I plated at some point to see if they are any good.
  • 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


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.

positive control for Cam/Spec
negative control for Amp
negative control for Kan

  • 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 2333:2343 2348:2343 2349:2343

To Do

  • microscope assays
  • transform and plate 2291 in MC1061 and MG1655.
  • transform 2291 into all 6 different strains.
  • depending on tomorrow, pour more 4 antibiotic plates.

Tahoura Samad 15:18, 21 June 2010 (EDT)

PDD Sugroup Work

  • 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.

negative control for amp.
positive control for Cam/Spec
negative control for Kan

To Do

  • pick and grow up cells in TB(for assay)
  • miniprep TIm's Ecoli
  • transform pick 2291 and grow up to miniprep.
  • transform 2331,2333, 2348 2349 into 2343 w BAC, 2343, 1934 and 2344

Tahoura Samad 16:09, 18 June 2010 (EDT)

PDD Subgroup Work

  • made competent cells of BAC with pBca1256-Bjh2344 in MG1655.

1. added 500 μL of fresh culture to 50 mL of Spec/Cam LB. Incubated for 3 hours in 37 shaker.
2. Chilled falcon tube.
3. Lit flame, poured from small erlenmeyer flask into falcon tube.
4. Chilled for 10 minutes.
5.Chilled rotor for an hour.
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.
7. Poured of supernatant.
8. Resuspended in 2.5 mL of ice cold TSS. <br. 9. Distributed into 98 μL aliquots.


  • electroporated pB6 into MG1655 with pBca1256-Bjh2343.

Protocol: Small Scale Electrocompetent Prep

  1. Grow up strain to saturation
  2. Innoculate 2YT with 1:30 of o/n. (add antibiotic to 2yt, and add about 5 mL of 2YT. (5 microliters of antibiotic)
  3. Grow uninterrupted for 4hrs @ 30C for ubercompetency. (30 C is the room next to the -80 freezer.
  4. Cool tube/flask; transfer 1.5 mL to chilled epi tube.
  5. Spin down for 20 sec at 14.1 rpm, discard supe, place immediately on ice.
  6. Resuspend with .5 mL 10% ice cold glycerol.
  7. Spin down for 30 sec (14.1 rpm), discard supe, place immediately on ice.
  8. Resuspend with .5 mL 10% ice cold glycerol.
  9. Spin down for 30 sec (14.1 rpm), discard supe, place immediately on ice.
  10. Resuspend in a small volume (100 ul) 10% ice cold glycerol.
  11. 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.
  12. Add ~2.5uL and 5 μLDNA to cells, pipette & stir to mix.
  13. 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.
  14. 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.
  15. stick it in the electroporator. voltage should be at 1600.
  16. press pulse twice. remove when Ch9 goes away.
  17. 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.

  1. Put in shaking 37oC incubator for 1 hr
  2. 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

  1. Grow recipient strain overnight in LB medium (2 mL culture is plenty).
  2. 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)
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)

3. mix 100 µL undiluted P1 lysate (from Josh's fridge)+ 100 µL recipient cells
4. Incubate tubes at 37 ˚C for 30 min.
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!)
6. Spin down cells
7. Resuspend each in 100µL 2YT/citrate.
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

  • On Monday, Eco/Bam Transfer the final PDD (2991)
  • On Tuesday, pick and grow up. Miniprep Tuesday evening or Wednesday Morning
  • On Monday, start transformations of PDD into competent cells.
  • On Tuesday, pick cells and grow up.
  • On Monday, pick and grow up 2343 if they grew.
  • On Tuesday, make competent cells of Bjh2343 with BAC.
  • On Monday, do something with Tim's Ecoli on plate
  • On Monday, check controls for competent Bjh2344.

Tahoura Samad 15:29, 17 June 2010 (EDT)

PDD Subgroup Work

  • 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.

BAC with pBca1256-Bjh1934 3 μL
BAC with pBCA1256-Bjh1934 1 μL and 1:10 dilution 1μL.

  • 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.
  • PCR looked good, with a band at 1 kb, which was the expected length for primers 25 and 26.
  • Picked ligation ecoli and grow up.Start time:10:40 am
  • make 2 1 L bottle of TB. TB is on the top shelf with the agar.Check back at 1:15 pmstored in cabinet by door e

To Do

  • On Monday, Eco/Bam Transfer the final PDD (2991)
  • On Tuesday, pick and grow up. Miniprep Tuesday evening or Wednesday Morning
  • On Monday, start transformations of PDD into competent cells.
  • 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.

Tahoura Samad 13:34, 16 June 2010 (EDT)

PDD Subgroup Work

  • check controls for competent cells for BAC with pBca1256-Bjh1934 in MG1655. Kan and Amp should be negative and Cam/Spec should be positive.
  • Kan and Amp were negative, Cam/spec was positive. looks good.

BAC with pBCA1256-Bjh1934 on Cam/Spec
BAC with pBCA1256-Bjh1934 on Amp
BAC with pBCA1256-Bjh1934 on Kan

  • 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.
  • none of these grew. at all.
  • 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
  • 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.
  • 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
  • streaked glycerol stock of BAC with pBca1256-Bjh2344 to grow up tomorrow and make competent cells.
  • miniprepped PDD in MG1655 and MC1061-(2331, 2333, 2348, 2349)

Ligation Ecoli Work

  • streaked glycerol stock of ecoli from Tim. Pick tomorrow.

Additional Notes

  • How to streak from glycerol stock:

1. dip tip of a p20 tip into glycerol stock
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.
3. take wire spreader and serially spread, flaming and ethanol between rounds.

To Do

  • Check on AK stuff (MC1061, Mg1655 at 5:00 pm, 5:30 pm. Miniprep those guys tonightDONE
    • Check on control for pB6 miniprep. (BAC with pBca1256-Bjh1934 cells in incubator)DONE
    • Pick 2344, and start growing up to make competent cells.
    • Pick ligation ecoli and grow up.DONE
    • make 2 1 L bottle of TB. TB is on the top shelf with the agar.DONE

Tahoura Samad 16:16, 15 June 2010 (EDT)

PDD Subgroup Work

  • 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.

  • Started competent cells for BAC WITH pBca1256-Bjh1934 (Medium Scale)

1. added 500μL of overnight-grown culture to 50mL Spec/Cam LB in an Erlenmeyer flask

  • 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.
3. Transfered culture to a 50 mL falcon tube that was sitting on ice.
4. Chilled overnight culture for 15 minutes to stop growth.
5. Got chilled rotor from cold room. (was in there for a couple of hours.) Changed rotor and rotor program.
6. Spun down cells at 4100 rpm for 13 minutes at 4 C.
7. Poured of supernatant and resuspended in 2.5 mL of ice cold TSS.
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.
9. Streaked for contamination on Amp, Kan and Cam/Spec. Should grow on Cam/Spec, but not Amp, Kan.
Plate names: iGEM control for competent cells BAC with 1934


  • Did genomic miniprep of Pb6.

Same steps as regular miniprep with the following changes.
This procedure is identical to the normal miniprep procedure except the step in red, and less culture is prepped
1. Pellet 1.5 mL saturated culture by spinning full speed, 30 seconds.
2. Add 250uL of P1 buffer into each tube. Resuspend the cells using
a vortexer.
3. Add 250uL of 2% SDS (stored in a 50 mL conicol)
(actually stored in falcon tube. Also, this step replaces the P2 step.
4. Incubate at 55 degrees until clear (roughly 15-20min?).
5. Continue as usual with N3 step.

  • 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)
1655-2333-(35 colonies)
1655-2348 (35-50 colonies)
1655-2349(35-50 colonies)

  • Picked two colonies of each (16 total) and am growing them up in a 24 well block in the 37 C shaker.

Gibson Work

  • diluted iGEM oligos 14-26 ( 5 μL oligo, 45 μL water)

Additional Notes

  • iGEM competent cell box 2 is in the same rack as iGEM competent cell box 1, in the back column.

To Do

  • 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
  • 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
  • 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
  • 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
  • streak out ligation e-coli.

Tahoura Samad 14:53, 14 June 2010 (EDT)

PDD Subgroup Work

  • 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.
  • picked pB6 so we can do a genomic miniprep on it and try to create BAC with pBca1256-Bjh2343 again.
  • picked BAC with pBca1256-Bjh1934 3μL so we can create a glycerol stock and competent cells.
  • put both in shaker upstairs to incubate overnight. start time: 11:00 pm
  • 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.
    • To make I M magnesium sulfate

-Added 12.4 grams of Magnesium sulfate to 50 ml of water in a falcon tube. Screwed on filter and vacuum filtered.

  • 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 4:20added 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

  • get strain out of freezer. Its in row B8. In -80 freezer, 2nd row from the top leftmost rack, in blue bottomed box.

Additional Notes

To Do

  • check on pBca1256-Bkh1934 and pB6.
  • Make glycerol stock
  • Make competent cells of pBca1256-Bjh1934
  • Genomic miniprep pB6
  • transform and plate pBca1256-Bjh2343 in MG1655(1) with pB6

Tahoura Samad 13:12, 11 June 2010 (EDT)

PDD Subgroup Work

PbCA1256-bJH2344 with 1 μL
PbCA1256-bJH2344 with 3 μL
PbCA1256-bJH2343 with 3 μL
PbCA1256-bJH2343 with 1 μL

  • 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.
  • 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.
  • 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
  • plated entire transformation on Cam Spec plates.
  • 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.

Creating Competent Lefty, Righty Cells, (MC1061 pir 1)

  • 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.

Tahoura Samad 16:56, 9 June 2010 (EDT)

PDD Subgroup Work

  • checked controls for competent cells. They looked good with no growth on Amp, Cam and Kan and growth on Spec.

Negative Control for Amp
Negative Control for Kan
Negative Control for Cam
Positive Control for Spec

  • 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.

Transformation of pBca1256-Bjh2343 in MG1655 with BAC
Transformation of pBca1256-Bjh2344 in MG1655 with BAC (left) ;Transformation of pBca1256-Bjh1934 in MG1655 with BAC (right)

  • threw plates away in red garbage
  • 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.
  • 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.

Assembly of iGEM parts 001 and 009

  • started miniprep of assembly 2 with Amy
  • 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)
  • the point of the gel purification was to get rid of parent vector and to select for the side of the vector we want,
  • 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.

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.


  • Ligation:
  • Set up ligation mastermix

Mastermix
6.5uL ddH2O
1uL T4 DNA Ligase Buffer (black-striped tubes)
0.5uL T4 DNA Ligase

DNA 6uL of L+R gel purified DNA
Added DNA to MM at 3:54pm. The ligation will be done at 4:30pm.


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

  • check on BAC with pBca1256-Bjh2343, BAC with pBca1256-Bjh2344, and BAC with pBca1256-Bjh1934 plates tomorrow.
  • pick colonies and grow up to make BAC -80 freezer stock


PDD Subgroup Work

  • Created -80 Stocks for pBca1256-Bjh1934 in MG1655, pB6 in DH5-alpha, MG1655, Bjh2343 in MG1655, Bjh2344 in MG1655r using this protocol
  • 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.

  • Did Miniprep of PB6 starting with 2mL of the overnight culture. Stored eluent (50 μL) of DNA in iGEM working box.

  • Made Competent Cells for pBca1256-Bjh1934 in MG1655,Bjh2343 in MG1655 and Bjh2344 in MG1655:

Recipe
1. added 500μL of overnight-grown culture to 50mL Spec LB in an Erlenmeyer flask for MG1655: (1:100)
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.
3. Put both flasks put on shaker in lab at 12:00
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.
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.
7. Removed falcon tubes from centrifuge and poured off supernatant into a bottle in the sink. Add bleach etc.
8. Resuspended pellet by vortexing in 2.5 mL of TSS, which can be found in the plate fridege, top right back.
9. Put on ice, and aliquoted 98 μL in 25 Eppendorfs for each of the four.
10. Put in -80 freezer. Third shelf? (or same shelf as JTK049, rack furthest to the rights.
11. Set up positive and negative controls for our competent cells

  • 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.

  • Transformed BAC into 1, 2, 3.
  • Plated on Cam, Spec plates.

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
  • make competent cells for pBca1256-Bjh2343 with BAC, pBca1256-Bjh2344 with BAC, pBca1256-Bjh1934 with Bac.
  • Find out what BAC (PB6) is
  • Look up what pBca1256-Bjh1934 is. (short description, part name)

Additional Notes

  • shaker on fourth floor is not at exactly 37, closer to 33, 34, so things take longer
  • make sure to scale down KCM by half for all the competent cells we made. (1, 2, 3, and 4)
  • BAC stands for Bacterial Artificial Chromosome.

Tahoura Samad 13:46, 8 June 2010 (EDT)

PDD Subgroup Work

  • all five transformations grew up well. The 2343 and 2344 appeared red.
  • 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.

Manual Assembly of iGEM10_13 and iGEM10_20

  • began manual assembly of assembly tree for iGEM10_13 and iGEM_20. (create iGEM10_008, 009, 014, 015, 016.)
  • set up Lefty and Righty digests and incubated: incubation should be done at 2:50 pm
    • Digested 8+4, 7+11 (x2), 9+20 (x2) with Lefty MasterMix.
    • Digested 9+3, 8+1, 7+2, 7+19, 8+12 with Righty MasterMix.
  • Zymoed the 8 digests.

  • Set up ligation reactions;
  • Ligate
    • Followed changed protocol (see 6/7/2010):
    • (Amy)Set up 5 ligations (made MasterMix recipe x6)

Mastermix
6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
0.5uL T4 DNA Ligase

DNA
3uL Lefty vector
3uL Right vector
Start time (4:15 pm, End time 4:45 pm)'


  • 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.

To Do

  • create glycerol stock of pb6 for -80 freezer. DONE
  • Create -80 stock for all 5 and competent cells for everything but pB6.
  • coat hangers? DONE

Tahoura Samad 14:15, 7 June 2010 (EDT)

PDD Subgroup Work

referenced Jin's notes:

  • (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.
  • 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)
4uL water
1uL of NEB2
0.5uL XhoI
0.5uL BamH1

Righty MasterMix (for 4uL miniprep DNA)
4uL water
1uL of NEB2
0.5uL XhoI
0.5uL BglII

  • R masterimix added to 7+23, 8+6, 7+5
  • L mastermix added to 9+8, 7+11, 9+20

  • Ligation:

Normal protocol with slight changes:
Mastermix Recipe
6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
0.5uL T4 DNA Ligase

DNA Recipe
3uL Lefty vector
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.
  • 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.

Additional Notes

  • Zymo can be used to of get rid of restriction enzymes. In this case, we can't heat kill because BamH1 survives.

To Do

  • Remind Tim to streak more of the MG1655 DONE.
  • Remember to pick the MG1655 colonies tomorrow. DONE
  • Pick and miniprep BAC from Germany DONE
  • Pick and miniprep 0343, 0344, pBca1256-Bjh1934 DONE
  • Bring folder for iGEM assembly trees. DONE
  • coat hangers?
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