iGEM 2018 Lab Notebook

Overview meeting.

Prepped some yPH500 yeast to grow overnight in the shaker.

Yeast grew successfully! Diluted ~500 mL of it into 2xYPAD and left to grow (and get happy) for ~4 hours.

Cut plasmid pKL1-354 with NotI to use for transformation of yeast cells & digested for 2 hours

Transformed yPH500 from yesterday with the linearized plasmid ( ​pKL1_354), rescued for 2 hours, and plated with hygromycin. Should now be yKL817 strain.

Golden Gate Assembly of pKL1-381 to pKL1-389.

Instead of making the master mix in the above protocol, we used NEB's GG master mix. Used ~150 ng of pKL0 and pYTK parts and ~30 ng of pKLE parts.

Grew up pYTK-013, pYTK-066, pKL0-007, pKL0-026, and pKL0-116 from glycerol stock. Will check them tomorrow morning.

Miniprepped bacteria glycerol stock cultures from yesterday. Restocked pYTK-013, pYTK-066, pKL0-007, pKL0-026, and pKL0-116 in freezer.

Checked pKL1 bacterial transformation plates:

Checked yKL816 transformation plates from Wednesday, 5/23. One plate has many colonies on it, the other has none. Placed both back in the incubator to grow further.

Later, both plates were taken out of the incubator. One still had no colonies so was tossed, while the other was kept on the bench to slowly grow at room temperature.

Today, retransformed pKL1-382, pKL1-383 and pKL1-385 through 389. See Thurs 5/24 for mixes.

Continued with the successful transformations from 5/24, pKL1-381, pKL1-382, pKL1-383, pKL1-384, and pKL1-385. Picked colonies and started growing the transformed bacteria in block. Block was left in 37 degree shaker overnight in preparation for miniprep tomorrow.

Picked colonies from pKL1-382, pKL1-383, and pKL1-385 plates (Plates for pKL1-386 thru 389 did not grow) & grew colonies in media until 5 pm

Miniprepped pKL1-381 through pKL1-385 from colonies grown up overnight from 5/29 & sent for sequencing at 5 pm

Miniprepped pKL1-382, -383, and -385 from colonies grown earlier & sent for sequencing at 7 pm

The two blocks used for these two minipreps were filled with media to prep for the growth of the remaining bacteria. These blocks were then kept in the refrigerator overnight

​Sequencing

The two blocks kept in the refrigerator overnight, one with pKL1-381 through pKL1-385 and the other with pKL1-382, -383, and -385, were placed in the 37 degree shaker at 9 am

Assembled pKL1-384, 386-389. Transformed and plated bacteria for each of these. Left them in the incubator to grow up over night.

Sent pKL1-381B, 382C, 383C and 385C off for forward-sequencing; these were the best sequencing results.

Threw out everything except for these plasmids.

Picked the new 381B, 382C, 383C and 385C media from the blocks, made glycerol stocks with them. Table below for future reference:

Picked pKL1-384, pKL1-388 and pKL1-389 into a block. pKL1-386 and pKL1-387 had no colonies. We suspect we weren't adding entry vectors to them during assemby.

Golden Gate assembly of pKL1-386 and pKL1-387. Transformed bacteria, plated them, and left in incubator overnight.

yPH500 overnight growth brought it to 1.69 OD. Diluted 100 uL into 10 mL of 2xYPAD. Left to grow for 4 hours.

Cut pKL2-226 with NotI to linearize it in preparation for yeast transformation.

Transformed the yeast into yKL814.

Picked colonies from the bacteria transformed with pKL1-386 and pKL1-387, grew them up, and miniprepped them.

yKL814 transformation was unsuccessful.

Re-digested pKL2-226 with NotI to prepare for a second transformation

Sent pKL1- 384B, 388A, 389A for forward sequencing, and 386A/386B, 387A/387B for reverse sequencing

Stickered pKL1-381, 382, 383, 385 and put them in the Jank Fridge with other pKL1s

Started growing yPH500 for overnight growth at 6:00pm

yPH500 overnight growth brought it to approximately 1.78 OD - too high!

Diluted 1:3 in YPD & grew for 1 hour during Khalil Lab group meeting (Current OD: ~1)

OD came out to be 1.4, so the yeast culture was trashed. Will try growing again tonight.

Assembled pKL2-233 to -238

Plated pKL1-334, 338, 339.

Plated pKL2-233 through 238.

Checked the yPH500 OD: only about 0.2 (9:30 am)

Picked colonies for pKL1-334, 338, 339 and pKL2-233 through 238 (pKL2-235 and -237 failed, -233 had only one colony), put the cells in appropriate media (LB+ Kan for pKL2 and LB+ cb for pKL1) in a block, and put in the incubator. (9:30 am)

Block with media with pKL1-336 and 337 (from 6/2) was placed in the incubator. (9:30 am)

Reassembled pKL2-233, -235, and -237.

Transformed bacteria with these pKL2's.

Assembled and transformed with pKL1-390 to -398

Transformed yPH500 with pKL2-226.

Made glycerol stock of pKL1-384, -386 through -389

(for -386 and -387, glycerol stocks were made for A and B. A and B for both of these numbers were forward sequencing)

Miniprepped pKL2-233, -234, -236, and -238 (both A's and B's)

Plated transformed bacteria (pKL1-390 to -398 and pKL2-233, -235, and -237) and transformed yeast (pKL2-226)

Checked on plates: transformed yeast (pKL2-226) and pKL1-393 failed. pKL1-397 only had one viable colony. pKL2-233, -235, and -237, pKL1-390 through -392 and pKL1-394 through -398 were successful.

Put the successfully transformed bacteria into two blocks (pKL2-233, -235, and -237 in one and pKL1-390 through -392 and pKL1-394 through -398 in another) into the 37 degree incubator for miniprep later.

yPH500 from last night had an OD of ~.9, so started to overgrow

pKL2-226 was linearized with NotI to prepare for yeast transfomation

Reassembled pKL1-393 and pKL1-397.

Transformed pKL1-393 and pKL1-397, left on bench to grow.

Sent pKL2-233, -234 (A + B), -236 (A + B), -238 (A + B), and -226 off for reverse sequencing.

Miniprepped pKL1-390 to -392, -394 to -398, pKL2-233, -235 and -237.

Accidentally put 250 uL of MX3 into pKL1-390A through -394B, then went back and put 100 uL MX3 in after the rest got 350 uL MX3. Hopefully that's okay.

Sent out for seqeuncing

Took out the yPH500 to yKL814 transformation: finally successful!

Found two blocks in the shaker that were left too long-- trashed

Found overgrown plate in the incubator-- trashed

Reassembled pKL1-391, 393, 397 as well as pKL2-233, 236

Transformed with pKL1-391/393/397

Transformed with pKL2-233, 236

Plated and placed in 37 degree incubator overnight.

Sent out for sequencing: pKL1-390A, 392A, 394A, 395A, 396B, 398A; pKL2-234B, 235, 237, 238B

Failure pKL1 - 393,397 & pKL2 - 233, 236

Success! pKL1 - 391

Block for pKL1-391 put in the frige for miniprep/sequencing/growing up stocks tomorrow

Transformed and plated pKL1-390-8

Transformed and plated pKL2-234, -5, -7, and -8

Reassembled, transformed, and plated pKL2-233, pKL2-236, pKL1-393, and pKL1-397

Put yPH500 in the shaker.

All successful plates except for pKL1-392 (lawn), -393 (all glowing), and -394 (lawn)

Took out yeast and started overgrowing

Picked colonies from the successful transformations (pKL1-390, 5-8 and pKL2-233-8) and placed in two blocks separating pKL1's and 2's

Transformed pKL1-392-4

Transformed yPH500 with pKL2-234, -5, -7, and -8.

Sent out pKL1-390-1, pKL1-395-8, pKL2-233-8 for sequencing

Plated pKL1-392-4

pKL1-393 and pKL2-233 failed (all glowed)

pKL1-391 sequencing came back-- failed

Growing up culture for glycerol stock (pKL1-392, pKL1-394, pKL2-236)

pKL1-391 and pKL1-383 reassembled (trying to fix pKL2-233 because -83 is specific to it)

Miniprepped pKL1-392 and -394

Sent out pKL0-6 (rev to check pKL1-393), pKL1-397 (fwd), pKL2-236 (fwd), pKL1-392 (rev), and pKL1-394 (rev) for sequencing

Transformed, plated pKL1-383 and pKL1-391, and placed in incubator for overnight growth

Checked pKL1-391 and -383: -383 failed (no colonies at all) and colonies were picked from -391 (A and B)

Growing cell culture of -391 A and B for miniprep and sequencing tonight

Plated pKL1-382 for overnight growth

pKL1-391A and B were miniprepped and sent for sequencing. Block filled with LB+cb media was put into the fridge for glycerol stocks to be made later.

pKL1-391A and B block was taken out of the fridge and was put in the incubator for glycerol stocks to be made

pKL1-391A and B glycerol stocks were made

pKL1-391A+B reverse sequencing failed. Threw out pKL1-391 glycerol stock.

Put pKL1-382A+B block into the incubator for glycerol stocks to be made tonight.

Assembled pKL2-242,3,4.

Transformed bacteria with pKL2-232,-3, and -4

pKL1-382A+B glycerol stocks were made

Sent out pKL0-234 and pKL1-382 A+B for reverse sequencing

Autoclaved pipette tips

Started growing yPH500 overnight

yPH500's OD at 9:30 was very high: about 1.4. Going to transform tomorrow instead

Picked colonies of pKL2-232,-3, and -4 from plate and put it in a block for miniprep and sequencing later

Miniprepped pKL0-125 (for restock and reverse sequencing) and pKL0-234 (for reverse sequencing)

Sent for sequencing: pKL0-234 A thru D, pKL0-125, and pKL2-242, 243, 244 A&B each

Grew overnight culture of yPH500 for yeast transformation attempt tomorrow

Grew overnight cultures for glycerol stocks of pKL2-242, 243, 244

Outgrew yPH500 in yPAD for transformation later

Made pKL0-234 and ran gel

Gel purification of pKL0-234

Transformed and plated pKL0-234

Checked yeast transformations (yKL854, -5, -6 with pKL2-242A+B, -3A+B, and -4B from Wednesday): no colonies. Left them in the incubator to hopefully grow up tomorrow morning.

pKL0-234 from Thursday night was successful: 5 colonies were picked and grown up in a block for miniprepping later.

pKL0-234 was plated on plain LB ("Chris Mancuso" <-> "CM" <-> "Chloramphenicol"). Redid the transformation, plated on chlor (really sure this time) and left to pick tomorrow morning.

pKL0-234 plate has "colonies" at 11AM

Turns out they're bubbles that spontaneously appeared in the gara.

Retransforming the pKL0-234 again.

Also put the plate back in the 37C incubator.

Checked yKL850s in the 30C. No growth; just dried media.

This time added 12 uL of 37ng/ul pKL0-234 (from gel extraction). 11:43 rescue start.

Plated transformation; let grow over the day.

At 6:30 took plates out of incu, left on bench

No growth on pKL0-234 plates.

Put yPH500 in YPD in shaker in the evening to grow over night.

Checked the yPH500 OD; found floating yeast in the YPD media, so threw it out.

Miniprepped pKL0-234 from Saturday.

Ran PIF3 on gel against pYTK001 to see if it was correct; PIF3 ended up being just the insert.

Assembled more PIF3 via PCR; we are running low.

Linearized pYTK001

Gel purified pYTK001

Made LB+Cm plates, YPD media

Assembled PIF3 + pYTK001 into pKL0-234

Put new yPH500 in shaker to grow overnight

Transformed, 30 min rescue pKL0-234

Plated pKL0-234

Checked pKL0-234 plates. Growth! All white colonies.

Picked 5 colonies from "new backbone" side and 4 from "old backbone" into 24-well block, 2 mL of LB+Cm at 9:50.

Started digestion on pKL2-242,3,4 at 11:45.

yPH500 overnight culture is growing (very slowly); we likely have not been picking enough cells from the original plate, an overcorrection from the fast growth observed earlier

yPH500 from yesterday is still growing slowly, likely due to high volume of media for a small amount of cells

Diluted yPH500 (1 mL) in 9 mL of 2xYPAD

Also, grew more yPH500 in 3mL of Meghan's YPD & 3mL of our YPD

Transformed yPH500 to yKL854-6

Miniprepped pKL0-234 A-H of Nikit backbone and A-I (not including E) of new backbone and sent for sequencing

Glycerol stocks were made of A-D of both backbones

Block being grown up for glycerol stocks to be made tomorrow (E-H for Nikit BB, F-I for new BB)

UPDATE

UPDATE

Yeast transformations yKL-854 to -6 failed (6/27)

pKL1-393 (failed -- 6/28)

Plates with pKL1-382 (6/28) with nikit backbone and pKL1-391 (6/26) with nikit backbone (NIK-C pKL0-234) were picked and grown up in a block for miniprep and glycerol stocks. (A, B, and C for -382 and A and B for -391)

(12:40) Grew up yeast colonies in a block (2 of our yKL814, 2 of Nathan's, 2 of our yKL817, 1 of Nathan's, and 1 of yPH500) (YPD+hygro for everything except yPH500 which was YPD)

(5:00) Grew up another set of yeast colonies

Miniprepped pKL1-382 ABC and -391 AB and sent it out for reverse sequencing. Growing up block again for glycerol stocks to be made in the morning

Growing up yPH500 for yeast transformation tomorrow

Performed our first eVOLVER experiment! 8 AM - 10:45 PM

Made glycerol stocks of pKL1-382 ABC and pKL1-391 AB -- waiting on sequencing to toss out bad ones

Restocking: grew up pKL0-006, pKL0-125, and pYTK-062 from glycerol stock in a block for miniprep later

yPH500 from last night was outgrown into yPAD

Linearized pKL2-242 through -6

t0=2:45 lights were turned on at 2:55pm

Turned off lights after 3 hours, t3=5:45pm

Transformed yPH500 to yKL-854 to -6

Miniprepped pKL0-006, pKL0-125, and pYTK-062

Assembled, transformed, & plated pKL2-233,-4, and -5

Our yeast strains tested yesterday in the eVOLVER experiment failed :( constit strains worked - all 817's working (although nathan's worked best)

pKL2-234A+B glycerol stocks were made

SD -ura plates were made

pKL1-391 assembled and transformed

pKL2-233, 234, and 235 were assembled and transformed

pKL2-233 and -4 failed

Colonies were picked and a block with pKL1-391 ABC and pKL2-235 AB was grown for miniprep and sequencing later

Put yPH500 in to grow overnight.

Digested pKL2-234,5,6,7,8. Check them at 12:45

yPH500 was at 0.5 OD. Picked it into 2xYPAD

Assembled pKL2-233,239,241. For pKL2-239 and 241, used all three pKL1-391 from yesterday (A, B, and C).

Transformed and rescued pKL2-233,239,241

Picked yPH500, yKL854,855,856 in YPD to grow overnight.

Picked a XYL-REG yeast colony (from Venkatesh) into SD -URA-TRP-HIS-LEU liquid media to grow overnight.

pKL2-233 failed; several colonies growing well, all green.

pKL2-241 with pKL1-391B and C failed; no growth.

pKL2-241 with pKL1-391A and all three pKL2-239 had multiple non-GFP colonies. These were picked into blocks to grow up over the day.

Streaked XYL-REG colony in liquid media on an SD -URA-TRP-HIS-LEU plate and left in incubator to grow.

Linearized pKL2-234,5.

Picked yKL854,855,856 into YPD (2:40 PM)

Miniprepped and grew up a block of pKL2-239 AA, AB, BA, BB, CA, CB and pKL2-241AA, AB

Did a fluorescein serial dilution and microbead serial dilution for the Interlab study.

Transformed the 8 E. coli NEB 5-alpha strains from reconstituted DNA for the Interlab study. Used our bacterial transformation protocol.

Made glycerol stocks of pKL2-239 AA, AB, BA, BB, CA, CB and pKL2-241AA, AB (first letter meaning the colony of pKL1-391)

Sequencing was redone on pKL1-391: all failed

pKL1-391 and pKL2-233 to be reassembled and transformed today

All interlab strains failed to transform.

Reassembled pKL1-391 to transform and plate.

Reassembled pKL2-233 with additional step for CIP to prevent entry vector from ligating back on itself.

Grew yPH500, yKL854, yKL855, yKL856 in YPAD to dilute & grow in YPD overnight & transform tomorrow.

pKL1-391 grew successfully! Picked colonies into a block for miniprep & glycerol stocking later.

pKL2-233 failed (both CIP and non-CIP)

Made new 2xYPAD and SD-URA liquid media.

Picked yPH500 into 4mL YPD in a culture tube at ~2:15 PM, to troubleshoot the yeast growth.

Picked 3 yPH500 colonies into our new 2xYPAD and old 2xYPAD to grow up in a block, in an attempt to troubleshoot the yeast growth.

Grew & Miniprepped pKL1-381, 391 D/E/F

Reverse sequenced pKL1 391 D/E/F

Picked and grew yPH500 in YPD, yKL854/855/856 in SD -URA at 5:00 pm in a block for yeast transformation tomorrow

Made temp glycerol for pKL391-D, E and F

Picked 100 uL of yPH500, yKL854 and yKL856 from the culture tubes into 6 mL of 2xYPAD. Left yKL855 in the YPD to grow up a little more. (~10:30 AM)

Growing more pKL2-234,5,7,8 from glycerol tonight; we're running low.

Linearized pKL2-234,6,7,8 with NotI for yeast transformation later today. In the incubator at ~11:20 AM, check them at ~1:20 PM.

Picked 300 uL of yKL855 to 2xYPAD (6 mL) at 12:30 PM

Plated pKLE-02 to see if backbone for pKL1-391 is wrong (should be all green tomorrow)

Sent pKL1-391DEF for fwd sequencing (rev failed but checking fwd in case it looks good/something is only wrong with the other end)

Picked another 3 colonies from the pKL1-391 plate, labeled G, H and I. Grew them up over the day and miniprepped them; sent them off for sequencing. (11 uL DNA, 2.5 uL primer, 1.5 uL water)

Transformed yKL867, 868, 870, 871, 873, 874, 876, 877, 878, 879, 880. Overnight rescue in 4 mL of YPD.

Plated yKL867, 868, 870, 871, 873, 874, 876, 877, 878, 879, 880.

Cut and ran undiluted stock pKLE-02 and pKL0-234 on a gel. Cut both with BsaI-HF at 37C for 1 hour.

Accidentally added Ethidium Bromide to the DNA samples instead of the gel; the gel run looked very jank but the sizes were correct on the ladder. Did not gel purify pieces.

Grew yPH500 and yKL854/855/856 overnight and outgrew in YPAD to transform later today.

Picked two colonies of pKL1-391 from the 7/4 plate. Picked 2 colonies from pKL1-391A, B, and P. Grew them up over the day, miniprepped them, and sent them off for sequencing.

Picked two colonies from each of the interlab controls and test devices into 4mL of LB+Chlor in cell culture tubes to grow up overnight.

Added 500 uL of each interlab colony sample to 4.5 mL of LB+Chlor. Checked the AB₆₀₀ and did a further 1:10 dilution into 4 mL total, as in the table below.

Took 300 uL of each diluted sample and read the AB₆₀₀ and fluorescence on the SpectraMAX plate reader. Data below.

Incubated all cultures for 6 hours, then took 300 uL of each sample and read the AB₆₀₀ and fluorescence on the SpectraMAX plate reader. Data below.

Diluted the two positive and negative control colonies down to 0.1 OD. In triplicate, diluted each of those 1:20 three times and then 1:10 twice. Plated 33 uL of the last three dilutions in each sample on one third of an LB+Chlor plate to grow overnight.

Cut pKLE-15 with BsmbI for ~2 hours. Gel purified half of the sample. CIP'd the remaning sample for 1/2 hour at 37C, did a PCR cleanup on it. Assembled and transformed 3 samples of pKL2-233: one with stock pKLE-15, one with cut pKLE-15 and one with CIP'd pKLE-15. Plated and let grow overnight.

Cut the stock pKLE-02 and diluted pKLE-02 with BsaI for 1 hour. Ran both on a gel (see below). Gel purified both samples; the diluted pKLE-02 sample had single-digit concentration, and was discarded. The gel purified stock pKLE-02 was used to assemble pKL1-391 and transform it into E. coli.

pKL2-233 had one colony, expressing GFP, from the cut pKLE-15 assembly. The stock pKLE-15 and CIP'd pKLE-15 had no colonies.

pKL1-391 had one colony. Picked it to grow up over the day for miniprepping at night.

Counted all of the colonies on all of the LB+Chlor plates. See below.

Assembled ​pKL1-391​pKL1-393​pKL2-233 using the ​Golden Gate Assembly and ​Golden Gate for pKL2's protocols.

Sent out pKL1-391 (from 7/25 assembly) for reverse sequencing.

Ran a 1:9 -> 1:9 serial dilution with microbeads on Peyton's chip; semi-successful.

pKL1-391 and -393 (from yesterday's assembly) colonies were grown up in a block and miniprepped. pKL2-234 and -235 were grown up from glycerol stock to restock DNA for yeast transformation

the pKL1-391 from yesterday's sequencing order failed

Grew up yeast for a yeast transformation tomorrow

pKL1-391's miniprepped yesterday failed reverse sequencing. pKL1-393 A, B, and C were successful. Sent for forward sequencing

pKL2-234 and pKL2-235 were linearized for yeast transformations

pKL1-393 sequencing came back: B was chosen

pKLE-02 was grown up for glycerol stock

pKLE-15 was miniprepped

Ran 1x Fluorescein solution through Peyton's serial dilution chip. The first run, the flow rates were accidentally bumped up in every step; the second run, the flow rates were the exact ones Peyton's math said they should be. We swapped runs based on "feeling" rather than timing.

pKLE-02 was miniprepped and cut. pKL1-391 was assembled and transformed

pKL2-233 was assembled and transformed

-391 had one colony and -233 failed

Tried different concentrations of cut pKLE-02 in assembly for pKL1-391. The concentration of cut pKLE-02 is 13 ng/ul and -391 was assembled with .25 ul, .5 ul, and 1 ul of the cut pKLE-02. -391 was also assembled with uncut pKLE-02 as a control.

Assembled and transformed pKL2-233 with 0.5, 1, 2, and 4 uL of gel-purified pKLE-15

Colonies were on all of pKL1-391 from yesterday's transformation. Several white colonies with the cut pKLE-02 and many colonies on the uncut pKLE-02 (more than half are white). Growing up 2 colonies from each of yesterday's assemblies of -391 (uncut (AA, AB), .25 ul (BA, BB), .5 ul (CA, CB), 1 ul (DA, DB)) and the one colony from 8/3's assembly (E) for miniprep and sequencing tonight.

Picked Nathan's yKL814, and streaked it on a YPD+Hygro plate. Did the same for our yKL814 B and E.

pKL2-233 failed again

Transformed yPH500, yKL854, 855, 856 with pKL2-234,235 to produce yKL 867, 868, 870, 871, 873, 874, 876, 877

Added a wash step with YPD, then split resuspended cells among 3 wells each with 3 mL of YPD in a block for overnight rescue

Cultured yKL814 A,B, E along with yPH500, yKL817 A, B, C, as well as two colonies of yKL814-NT from a plate in the cold room and 4 colonies of yKL814-NT from Nathan's block

Plated transformed strains from previous day and incubated until Saturday, 8/11

White light experiment with our yKL-814 and -817 and Nathan Tague's -814 (both from plate and media)

t1: 10:50

t2: 11:50

t3: 1:30

t3: 2:40

t4: 3:40

t5: 4:40

t6: 5:40

t7: 6:40

Assembled and transformed pKL2-233 as pKL2-233 E15/383, E15/3KL, EKL/383, and EKL/3KL, where KL denotes a piece of DNA that was cut with the Khalil lab's stock of enzyme rather than our own

Also assembled and transformed pKL2- 239, 240, 241

pKL2-233 grew! E15/3KL, EKL/3KL, EKL/383 had several colonies, which were picked and grown for sequencing

Sent for reverse sequencing: pKL2-233 E15/3KLA, EKL/3KL A&B, EKL/383 A&B, pKL2-239 A&B, -240 A&B, -241 A&B

pKL2-233's sequencing failed

Reassembled pKL2-233

Last round of yeast transformation failed, except for yKL870 and yKL876, which were re-struck and grown overnight

Grew overnight liquid cultures of yPH500 and yKL854/855/856 for another round

pKL2-239 through -241 A's and B's came back from forward sequencing-- all good. A's were chosen.

Forgot to make glycerol stocks of these pKL2's -- transforming with pKL2-239 through -241 today

pKL2-233ABC were grown in a block for miniprep and sequencing later

Transformed for yKL 867/868, 870/871, 873/874, 876/877, along with yKL 851/852/853

Added a wash step of YPD after transformation, and rescued in 250 uL of YPAD for 2 hours (split resuspended cells across 2 wells each)

assembled pKL1-399, 400, 401

pKL1-399 has one colony; picking to grow up for miniprep. 400 and 401 have no colonies.

pKL2-239,40,41 from yesterday's transformation had numerous white colonies. Picked one at random from each, and put in a block for glycerol stock

At 5:00, began the ​Running the eVOLVER protocol. Spiked in the various yeast strains in the following pattern:

The next morning (~7:30 AM) vials 12, 13, 14 and 15 were overgrown (OD ~1.5). After checking the experiment protocol, they weren't given an upper OD threshold (set to 9999). Still taking readings anyway, since bad data is better than no data.

The FACS plates are laid out as follows:

Grew overnight cultures of yKL867/870/873/876/877/878/879/880 along with yPH500 for Red Light Preliminary Experiment on Tuesday, 8/21

Also grew overnight cultures of yPH500, yKL854/855/856 for another round of transformation along the same protocol we have been following (overnight culture of parent strains in 4 mL of appropriate media in culture tubes; Outgrowth in a sterile block of 400 uL in 2 mL of YPAD; Linearization of DNA used NotI for a shortened time of 1 hour. transformation using the PEG/LiAc/SSDNA with an overnight YPD rescue)

Reassembled pKL1-381 and transformed into E. Coli

Performed Red Light Experiment, 7 hours of induction of strains with red light according to the following table

Transformed yeast cultures from Monday, 8/20 and did an overnight YPD rescue

Grew and Miniprepped pKL1-381 A&B but forgot to make glycerol stocks

Grew overnight cultures of yPH500, yKL854/855/856 to prepare for another round of yeast transformation following a different protocol (overnight culture of parent strains in 10 mL of appropriate media in flasks, beginning at 3 PM; Outgrowth in flasks, in 3 mL of YPAD per transformation, using volume of cells equal to 10% of the YPAD volume; Linearization of DNA used NotI for the normal 2 hours. transformation using PEG/LiAC/SSDNA method with an appreviated 1.25 hour rescue)

Plated rescued yeast from Monday, 8/20, on YPD + Hygro plates. Checked OD >= 1.5 for all strains; It is likely that the cells are progressing too far into stationary phase & thus do not survive when plated

Transformed yeast from Tuesday, 8/21, and performed an abbreviated 1 hour 15 minute rescue in an effort to prevent the cells from progressing too far into stationary phase. Plated rescued strains on YPD+Hygro plates marked "Flask"

Reassembled pKL2-233 as pKL2-233 A & B using pKL1-381 A & B (Important: Removed a step in the thermocycler golden2 protocol that held the sample at 55C for 10 minutes; This step likely allows the BsmBI to cut up pKL1-382, which has internal BsmBI cut sites in the PIF3-NLS sequence. These sites may ligate back together but may not leave a scar, which may be the source of our difficulties in transforming pKL2-233)

Transformed pKL2-233 A&B to sequence and make stocks alongside pKL1-381 A&B to make glycerol stocks

Transformations were successful! Put plates in the cold room to pick colonies later for sequencing and glycerol stock making.

Growing up pKL2-233 BA,BB, and BC and pKL1-381AA, AB, BA, and BB for miniprep and sequencing later today

Miniprepped and sent for reverse sequencing. Growing up overnight for glycerol stocks in the morning

Assembled pKL1-399, 400 and 401. Used pKLE-22 for pKL1-399 and pKLE-07 for 400 and 401.

Transformed TG1 comp cells with the above assembly, plated on LB+Cb.

Picked two colonies from pKL1-399, -400, and -401 each and grew them up. Miniprepped, sent for sequencing, and started growing them up for glycerol stocks in the morning.

Failed yeast transformation

Glycerol stocks of pKL1-399, -400, and -401 (A's & B's) were made.

pKL1-399 and -400 were successful: pick 399A and 400B. pKL1-401 failed.

Reassembled, transformed and plated pKL1-401.

Reassambled, transformed and plated pKLE-15

New pKLE-15 has no green colonies.

Yeast transformation

Sequencing came back for pKL1-399 to -401: choosing 399A, 400A, and 401A.

Retransformed and plated pKL1-401A for use in glycerol stock tomorrow.

Assembled pKL2-245.

Transformed with pKL2-235

Assembled and transformed with pKL1-383

Made glycyerol stocks of pKL1-401

Picked colonies from pKL1-383. pKL2-245 transformation failed (one green colony)

Miniprepped and sent pKL1-383A and B for sequencing

For pKL2-235: put water and DNA mix into freezer, did not assemble

Assembled, transformed, and plated with pKL2-235

Started yeast cultures for eVOLVER experiment on Wednesday

made glycerol stocks of pKL1-383A and B

pKL2-235 from 9/9 assembly failed

Yeast Transformation

Plated from yeast transformation's rescue this morning

Started red light eVOLVER Experiment #1:

t0= 10:40am

t1 = 11:40 am

t2 = 12:40 pm

t3 = 1:20 pm

t4 = 3:20 pm

t5 = 4:20 pm

t6 = 5:20 pm

t7 = 6:20pm

t8= 7:20pm

Resuspended the 1000 ng DNA samples from IDT (vioC-GG, vioD-GG and xylA-Clos) in 50 uL of water.

Assembled pKL2-245 and pKL0-237, 238 and 241. For the latter three, used 1 uL of DNA insert and 1 uL of precut pYTK001 backbone. Accidentally made two copies of pKL0-237, 'Dupe A' and 'Dupe B.' Transformed and plated all of these assemblies.

pKL2-245 has a handful of white colonies and one green colony. Both duplicates of pKL0-237 look normal (50/50 green/white) and pKL0-238 and 241 have no colonies. Picked the successful colonies into a block for miniprepping tomorrow.

pKL1-383 forward sequencing came back: success! A will be chosen

Miniprepped pKL2-245 and pKL0-237 samples from yesterday. Sent off for sequencing.

Assembled pKL2-233, pKL0-238 and pKL0-241. For pKL0-238 and 241, two samples of each were assembled: one with 2uL of insert, one with 5 uL of insert. Ran out of ddpYTK001 afterwards, and was not able to re-run a 1uL insert assembly.

Restocking pYTK001 from gylcerol stocks in a block overnight.

pKL2-233, pKL0-238, and pKL0-241 transformations failed

pKL2-233 was reassembled

Miniprepped pYTK001 from the blocks.

pKL2-233 transformation was successful!

pKL2-245 sequencing for both A and B came back fine; chose A. Forward sequencing for pKL0-237 AA was a bad read, and pKL0-237 BB has a noisy sbp mutation at 1323 (G->A).

pKL2-233 sent for sequencing along with pKL0-237 AA, BB re-do

Overnight cultures of yPH500, yKL 857,858,859,867,870,873, and 817 grown for a red light experiment tomorrow

2 mL YPD spiked with colony

Dilute 60/40 every hour

Ran 5 ​Precut Digest's on the pYTK001 from Friday. Gel extracted. Very low concentrations; scrapped it.

Around 11:00 AM, picked four new colonies from the same pKL0-237 plate, denoted pKL0-237 AC, AD, BC and BD. All were picked into LB+Cm. They should be ready to miniprep at 7:00 PM.

Digesting 4 sets of pYTK001 again. This time, the mix is 12.5 uL of DNA (~0.15 ug/uL concentration across the board), 3 uL BsmBI and 4.5 uL of Buffer 3.1. All of them went into the 55^oC incubator at 3:50 PM.

Additionally, two more pYTK001 cultures were started from glycerol stock at 4:00 PM in plain LB. This is a mistake, and should have been done in LB+Cm.

Took first time point at t0 = 10:15 AM

Took second time point at t1 = 11:15 AM

The lamp was not turning on after t1 was taken; after some troubleshooting, got it working again. Induction began again at 11:50 AM.

Took third time point at t2 = 12:15 PM

Took fourth time point at t3 = 1:15 PM

Took fifth time point at t4 = 3:15 PM

While diluting after t4, accidentally added 800 uL of YPD+Hygro to the yPH500 wells. Promptly removed 800 uL of media from those two wells, and added 800 uL of YPD. Expect low cells counts.

Took sixth time point at t5 = 4:15 PM

Took seventh time point at t6 = 5:15 PM

Took eighth time point at t7 = 6:15 PM

Miniprepped pKL0-237 AC, AD, BC, and BD

Glycerol stocks for pKL0-237 AC, AD, BC, and BD were made

Yeast transformation.

pKL0-237 BD has a sbp deletion at 1802 bp, roughly 600 bp into the read. The rest look good.

Assembled pKL0-237, 238 and 239 with both precut ddpYTK001 and uncut pYTK001 backbones. Used 4 uL of 50 ng/uL gBlock for all of the inserts, 1 uL of 5 ng/uL ddpYTK001 for the precut run and 0.4 uL of 150 ng/uL pYTK001 for the uncut run. Otherwise, followed ​Golden Gate for pKL2's to the letter. Left assembled DNA on the bench overnight.

Transformed and plated pKL0-237, 238, and 241.

PWM experiment 1 with different light pulsing patterns in LOV2

Assembled & transformed pKL1-402, 403, 406, 407, 410, 411

Transformation failed

Reassembled pKL1 - 402, 403, 406, 407, 410, 411 & transformed

Transformation failed again - hadn't been adding entry vector to assembly & thus transformants did not have Carbenicillin resistance

Reassembled pKL1-402,403,406,407,410,411 with entry vector this time

Regrew pYTK001, pKL0-107 overnight for restocking

Transformation was successful! Picked 2 colonies each of pKL1-402,403,406,407,410,411 to sequence & assemble in pKL2s later

Miniprepped pYTK001 & pKL0-107. Obtained good yields; hopefully this prep of YTK001 is not bad like the last

Reassembled pKL0-239, 241 with the new prep of pYTK001 (1 uL) and respective inserts (1 uL), using a 30 minute pre-cut at 55C

In preparation for making biobricks, at approx 8:30 PM, picked pKL1-299, 381, 382, 383, 386 and pKL2-226, 233B and 238 from glycerol stocks into a block, 2 wells apiece. Looked for pKL1-351 but could not find it. Threw pKL2-233A out after checking the sequencing and seeing 233B came back good, and A bad. Saw that there was a pKL1-383 from May(?) and a pKL1-383 A&B set from 9/9/18. Picked the earlier pKL1-383 into the block; later realized the dupes. Will doublecheck tomorrow.

Miniprepped and spec'd pKL1-299, 381, 382, 383, 386 and pKL2-226, 233B and 238 from yesterday. Prepared a 6ug, 50 uL double digest. For pKL1-299, 381, 382, 383, 386, used XbaI and SpeI as per BioBrick format. Ran out of SpeI after those, and digested pKL2-226, 233B and 238 with XbaI and PstI-HF, will adapt the backbone digest for these. Additionally, left one sample each of pKL2-226, 233B and 238 with appropriate volumes of water, DNA and CutSmart buffer for later digest with SpeI.

Started growing 2 colonies of yKL814, 1 of yKL817 and one of yPH500 into liquid media for Saturday's eVOLVER experiment.

Took digest out of the incubator at 1:30 PM.

Most, unfortunately, seem to have run off into the wells, or gotten stuck in them. pKL1-386 and pKL2-226 look good, so gel extracted those.

Around 8:00 PM, spiked cultures from the morning into the eVOLVER and left to grow overnight. See map below.

Started eVOLVER light induction at 1:45 PM.

vial 6 is dead; the OD decreased overnight.

t4, vial 1 is contaminated; do not use it.

At 6:20, noticed the eVOLVER had not been diluting for over an hour. All vials then diluted down to <0.1 OD. Experiment was aborted.

At 7:30 PM, spiked yPH500, yKL857A and yKL857B (as defined by the 9/25 shaker experiment) into 10 mL YPD in flasks and yKL817, yKL814 rep1 and yKL814 rep2 into 10 mL YPD+HO in flasks. These are for the eVOLVER experiments tomorrow. The two yKL814 replicates are from different colonies on the "Nathan restruck" plate from 8/7. Put all flasks in the 30^oC shaking incubator.

Restruck yKL857A and yKL857B onto their own SD-URA plates. Put in the 30^oC incubator.

After many trials and tribulations, the two eVOLVER experiments were called off.

Digested the 38 uL of 100 pg/uL pSB1C3 with XbaI and PstI-HF overnight.

Double-digested, ligated and dried every biobrick we shipped.

Grew up relevant strains for tomorrow's experiments (see tomorrow's maps)

Red and blue step experiments were done in the eVOLVER (Crick)

Red pulse experiment was run on eVOLVER (Darwin)

Red light orthogonality experiment was run in the shaker. (yPH500, yKL814, yKL817, yKL857)

Ran all of the plates from yesterday's experiment and the blue light orthogonality experiment in the BUMC Flow Cytometry core's machines.

Grew up strains for tomorrow's blue pulse experiment.

Ran blue pulse experiment in Darwin

Plated vioABE+LOV2->vioC, vioABE+pTEF1->vioC, and vioABE on two plates apiece. One of each plate was grown up on the bench under a blue light, one of each plate was grown up on the bench in total darkness.

After red orthogonality failed to induce in the shaker, ran a follow-up experimentin the eVOLVER

The blue light vioABE+LOV2->vioC strains are pink and the same strains in darkness are green! Everything else died.