Berk2010-Tahoura

June

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.