Berk2006-ConjugationTeam

JL_06/26/06 Today we did the TraJF/oriTF experiment. We Conjugated EC100D:pr+ with:

1. TlambdaOlambda+1064
2. TlambdaOlambda+1093
   

3) TlambdaOlambda+1064+1093
4) DH10B/1064
5) F plasmid on Kan
The Conjugation process we used is as follows:

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)