Berk2010-Tahoura

June

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.
• 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.

IBB Work

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

To Do

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

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

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

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

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

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

To Do

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

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

PDD Subgroup Work

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

Life Act

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

Other

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

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

PDD Subgroup Work

• nothing

LifeAct

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

• will miniprep in the afternoon.

Other

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

To Do

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

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

PDD Subgroup Work/Discussion With Jin

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

---

Minimal Media Testing

• Day 1

1. Streak out 3, 5
2. Pick and grow up in TB with 30 mM MgS04 (30 μL per mL) overnight.(16 hrs) Do two colonies of each 3 and 5.

• Day 2

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

Lifeact

• ran colony PCR for 32 colonies.

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

NLS

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

To Do

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

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

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

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

• most of my transformations with the new competent cells were successful.

Competent cell	Number	PDD	Growth in initial competent cells	Growth in Small Scale Competent Cells	Result of Assay with Jin		Conclusion
pBca156-Bjh2343	1	2331	0	tons of colonies	NOT DONE		transform new, assay
pBca156-Bjh2343	1	2333	0	tons of colonies	NOT DONE		transform new, assay
pBca156-Bjh2343	1	2348	1 colony (twice)	tons of colonies	NOT DONE		transform new, assay
pBca156-Bjh2343	1	2349	0	50-100 colonies	NOT DONE		transform new, assay
pBca156-Bjh2343	1	2291	0	100-200 colonies	NOT DONE		transform new, assay
pBca156-Bjh2343 with BAC	7	2331	0	15 colonies	NOT DONE		Today
pBca156-Bjh2343 with BAC	7	2333	0	2 colonies	NOT DONE		Today
pBca156-Bjh2343 with BAC	7	2348	0	4 colonies	NOT DONE		Today
pBca156-Bjh2343 with BAC	7	2349	0	0	DEAD		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2343 with BAC	7	2291	0	0	DEAD		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344	2	2331	0	tons of colonies	NOT DONE		make new, transform, assay
pBca156-Bjh2344	2	2333	0	tons of colonies	NOT DONE		make new, transform, assay
pBca156-Bjh2344	2	2348	12 colonies	TO DO	V (UNSTABLE)		make new, transform new and old, assay
pBca156-Bjh2344	2	2349	6 colonies	TO DO	V V		make new, transform new and old, assay
pBca156-Bjh2344	2	2291	7 colonies	TO DO	V V		make new, transform new and old, assay
pBca156-Bjh2344 with BAC	6	2331	tons of colonies	X	X		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344 with BAC	6	2333	tons of colonies	X	X		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344 with BAC	6	2348	1 colony (twice)	100-200 colonies	NOT DONE		Today
pBca156-Bjh2344 with BAC	6	2349	1 colony(twice)	35-50 colonies	X		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh2344 with BAC	6	2291	35-50 colonies	100-200 colonies	V V V		transform new, assay
pBca156-Bjh1934	3	2331	35-50 colonies	X	X		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934	3	2333	<100 colonies	X	X		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934	3	2348	tons of colonies	X	V V		transform old, assay
pBca156-Bjh1934	3	2349	tons of colonies	X	V V		transform old, assay
pBca156-Bjh1934	3	2291	tons of colonies	X	V		transform old,assay
pBca156-Bjh1934 with BAC	5	2331	tons of colonies	X	X		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC	5	2333	tons of colonies	X	X		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC	5	2348	tons of colones	X	X		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC	5	2349	1o colonies	X	X		\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
pBca156-Bjh1934 with BAC	5	2291	tons of colonies	X	V V V		transform old, assay

• competent cell checks for new 1 and new 6 looked good.
• miniprepped IBB as CA lefty, CK lefty and CK righty, and sent out for sequencing with ca998.

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

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

Kan
LB
Cam
Amp
Spec

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