Berk2006-ConjugationTeam

SIL_06/27/06

Results from 6/26/06 OriT/TraJ Experiment

Culture	OriT	TraJ	TriK	TriA	Comments
1	+	-	6	2	looks good
2	-	+	7	n/a	missing pBad promoter
3	+	+	3	5	missing pBad promoter
4	+	-	n/a	4	looks good
5	+	+	>1000	n/a	looks good
6	+	-	6	200	wierd, hoping for a smaller TriA count, but possible
7	+	+	3	1	possible tube switch with 9
8	-	+	0	n/a	looks good
9	+	-	n/a	>1000	possible tube switch with 7
10	+	+	>1000	n/a	looks good

--Samanthaliang 14:40, 27 June 2006 (EDT)

SIL_06/26/06 Today we did the TraJF/oriTF experiment. We Conjugated EC100D:pr+ with:

1. TlambdaOlambda+1064 (plated on TriA and TriK)
2. TlambdaOlambda+1093 (plated on TriK)
3. TlambdaOlambda+1064+1093 (plated on TriA and TriK)
4. DH10B/1064 from -80 (plated on TriA)
5. F plasmid on Kan
   (Plated on TriK)
6. Rlambda+1024 (Plated on TriA and TriK)
7. Rlambda+1024/1001 (Plated on TriA and TriK)
8. Rlamdbda+1001 (Plated on TriK)
9. DH10B/1001 from -80 (Plated on TriA)
10. wt RP4 (Plated on TriK)
    

Also, we should note, that the TlambdaOlambda+1064 and the DH10B/1064 cells were the only ones that were really red. Rlambda+1024 was a peachy color. TlamdbdaOlambda+1064_1093 and Rlambda+1024/1001 were both a very very faint shade of pink, which is troublesome, since they should have come out red as well.
We also mini-prepped 4 clones of pSB1A2-J23003(TraJr-TT)and created an APE file for it. We had to also sequence them because mapping was not possible because the distinguishing fragment from pSB1A2-J01001, which J23003 was constructed from, the terminator was too small of a sequence and no enzymes could be used to cut so that there was an obvious difference between the two plasmids when mapped.
Lastly, the conjugation experiment with Rlambda and RP4-G that we did on Friday worked very well. In fact, it was too efficient and ended up as a dense lawn. Thus, we restreaked the plate, but only after diluting it from 1uL to 1mL LB twice, then taking only 24uL of the diluted cell solution and plating it.
--Samanthaliang 17:08, 26 June 2006 (EDT)

JL&SIL_06/22/06
Today we mapped pSB3C6-J01024 using EcoRI and SpeI and pSB3C6-J01093 BamHI and SpeI. We had 100% recovery of 4 clones of each, and did not get any colonies of just the backbone alone. This was determined by a analytic digestion which resulted in 2 stripes for each of the 8 columns we ran in the gel (if it had only been 1 stripe, that would've indicated the colony was that of the the backbone pAC997). Because of these positive results, I made -80 stocks of the clone 1s of both pSB3C6-J01024 and pSB3C6-J01093, and put the minipreps of clone 1 and 2 of each in the freezer.
(ladder, 4 clones of J01024, 4 clones of J01093)
We also digested, gel purified, ligased J01001 and B0015, and transformed by heat shock to get the product J23003 (TraJf-tt).
We also replated RP4 and F on KAN plates, EC100D:pir+ on TRI plates, and 1064 and 1001 on AMP plates. Then we picked colonies from the Rlambda plate from 6/19/06 to grow into competent cells.
Lastly, we are preparing for a big experiment regarding the TlambdaOlambda cells, by transforming plasmid pSB1A2-J01064 into one sample, plasmid pSB3C6-J01093 into another sample, and both of those plasmids into a third sample (this is the one we are most concerned about) using heat shock.
The purpose of this experiment would be to determine whether or not the F plasmid with the KAN marker, J01064 with the AMP marker and OriT, and J01093 with a CAM marker and TraJf will transform into a new plasmid with a TRI marker.
--Samanthaliang 20:11, 22 June 2006 (EDT)

SIL_06/19/06
Today we subcloned pSB1A2-J01024 into the pAC997 backbone to create the final product pSB3C6-J01024. We did this by digesting pSB1A2-J01024, gel purifying it, ligating it, and then heat shocking it. We also subcloned pSB1A2-J01093 into the pAC997 bacbone to create pSB3C6-J01093, starting from a PCR product of J01093 because in a previous experiment J01093's results came out unclean. This was also done through digestion, gel purification, ligation and heat shock.
We also restreaked Rlambda from the -80 freezer stocks onto a Kan plate.
Lastly, we made sure that J01001 was in the freezer for future use.
--Samanthaliang 19:17, 19 June 2006 (EDT)

JCA_06/17/06
We had some difficulty with the traJF and traJR stocks for our initial tests of conjugation. It is unclear what plasmid the -80 stocks are inserted into. We tried to grow up the "J01024" from Box2>7v4 in carbencillin media. It didn't grow. Yesterday we grew up the "J01024/KAN" in Box1>9v1. It grew just fine and the cell pellet was slightly pink due to its Ptet-RFP cassette. It is unclear whether this is pSB1AK3 or perhaps a pSB*K* plasmid? Unclear, but we're going to transfer it into pSB3C6 anyway, so who cares. In addition, I was concerned that the oriTF plasmids might have CmR markers on them. The biobricks annotation for the J01002 from the plates indicated it was pSB1AC3. That would be bad--it needs to be just ampR. So, we grew up the two different J01002's from the freezer stocks (Box1>6v4) and (Box1>8v2). The second one for some reason is not in the slot indicated on the -80 stocks page. Someone will need to re-update the -80 stocks page. Dan spotted the two J01002 strains and J01064 from Box1>5v1 (oriTF/OnRFP). All three grew spots on amp but not on Cm or Kan. So, they are all 3 on pSB1A* plasmids, so it's all good. Incidently, J01064 was flaming pink in cell pellets. Much brighter than J01024 despite them having the same RFP cassette. I miniprepped J01024 and J01064.

JL_06/07/06
Goal: We want to determine the antibiotics resistence of the F and R knockout plasmids (TlambdaOlambda)
Results:
We grew TlambdaOlambdaRlambda to early log density. We then streaked the cultures with pipette tips on LB agar plates containing the following anti-biotics:

1. 15ug/mL gentamicin (G)
2. 50ug/mL ampicillin (A)
3. 25ug/mL chloramphenical (C) ---errr, axe that datapoint, the plate disapeared.--JCAnderson 13:45, 8 June 2006 (EDT)
4. 50ug/mL spectinamycin (Sp)
5. 25ug/mL tetracycline (Tet)
6. 20ug/mL trimethylprim (Trim)
   

TlambdaOlambda grerw on kan only
Rlambda grew on kan and tet only--JCAnderson 19:07, 7 June 2006 (EDT)