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

Today we redid the experiment, using Arg. with the F plasmids for the Pbad promoter, and the R plasmids the same as before, in order to see if tube 7 and 9 really were switched.
The diluted restreaking of Rlambda and RP4-6 from yesterday failed and a lawn was still achieved, so we redid that again this time, adding another 10 fold dilution. We also poured more TriA and TriK plates.
Also we streaked all of the plasmid combinations (1-10) from the OriT and TraJ experiments onto a Kan plate, an Amp plate, and a Tri plate, in order to make sure that all the plasmids have the antibiotic resistances that we think they have, and that they don't have any extras.
--Samanthaliang 14:41, 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)