Berk2006-ConjugationTeam2

Return to main page

JCAnderson 20:37, 16 September 2006 (EDT)

I ran the gel on the EcoRI/PstI pBca1020-J23038 mapping, all 4 look the same (and are correct). Clone 1 is a little dirty, so we'll proceed with clone 2.

JL-09/16/06

Todays's to do list:

1. Mini, map pBca1020-J23038 -- map with EcoRI/PstI to confirm the size of the smaller fragment
2. Mini, stock, send for sequencing J23077 (with ca1000) and J23078 (with ca998) -- miniprep 2 of each (assuming the cell pellets are white, not pink) and send 1 of each for sequencing.
   

I mini-preped clones #1 and 2 of each

1. restreak on Kan, grow at 43 degrees J23015 x pCP20
2. Make a -80 stock of the J23069 culture (traG, call it J23057) and do a genomic miniprep
3. set up a PCR pf J23069 with your traG 20mers

JL_09/13/06

Todays's to do list:

1. submit the new clone of J23072 for sequencing with ca1000
2. culture pcrs for J23074 and J23075
3. electrocompetent cells of TraG Ko (to transform with pcp20)

JL_09/11/06

TOday's to do list:

1. dilute & replate J23015 on kan at 43 degrees.
2. make competent cells of trbC
3. make Cmr plates
4. restreak J23069 clone #2 onto Cmr plate

JL_09/08/06

TOday:

1. Mini, map, seq pSB1AK3-J23075 trbC complementation
2. Mini, map, seq pSB1AK3-J23074 TraGr complementation
3. J23015 (OriTf::Cm), Transform pCP20, plate on Amp 30 degrees.

JL_09/06/06

There were no colonies on trbC KO #2 conjugated with EC100D::pir+ on TriK and TriC.
To Do list:
Grow to Mini, map, seq pSB1AK3-J23075 trbC complementation (later move to pSB3C6)
Grow to Mini, map, seq pSB1AK3-J23074 TraGr complementation (later move to pSB3C6)
Restreak J23015 (OriTf::Cm) (later transform pCP20 with an amp neg control, then pCP20)
Restreak J23016 (trbC::Cm) (later transform in pSB1AK3-J23075, mate with Ec100D)
PCR with JL16/JL17 genomic J23016 (trbC::Cm) (low priority)
Screen J23017 (traG::Cm) restreak to comp
Today I grew up four cultures each of pSB1AK3-J23075 and pSB1AK3-J23074, restreaked J23015 from the -80 stocks, grew up sat. culture of J23015 and J23016, and ran a gel of the 3 PCRs of J23069.
The pic of J23069 (lane 1=clone3, lane 2=clone2, lane 3=clone1, lane 4= ladder)

J23069 is supposed to be around 1694 bp long. Therefore, clone 2 is correct.

JL_08/25/06

The conjugation experiment worked! J23015 x J01064 with EC100D::pir+ on TriK had no colonies, but on TriA there were >1000 colonies.
I ran a gel of trbCf KO (4 clones) and J23069 (3 clones). All of the trbCf KOs were correct (around 1400 bp), but there were no bands for the J23069 clones. Therefore, I am doing another PCR of J23069, but this time I'm using the 4K45 program (with DMSO). Also, I'm conjugating trbCf KO #2 with EC100D::pir+ and plating it on TriK and TriC. We expect to see no colonies on both plates.

JL_08/24/06

Today, I did a mini-prep of OriTf KO (4), trbC KO(4), and J23069 (3)(TraGr KO). I also PCR-ed them. I used the oligos JL3 & JL4 for OriTf KO, JL16 & JL17 for trbC KO, and JL18 and JL19 for J23069. I conjugated J23015 x J01064 with EC100D::pir+ and plated them on TriK and TriA.

MF_08/18/06

Today the OriT(J23015) and trbC(J23016) knockouts grew after I re-did the electroporation yesterday. So, next week Sam or Jen will transform the knockouts with pCP20 so it will recombine with the CAM gene and just leave 1 frt site. Then we will heat treat it to get rid of pCP20 and then mate with EC100D::pir+.
Also, the plate of the 10^6 fold dilution of J23069 grew, so on tuesday Sam will grow it up, and Jen will transform it with pCP20 and then mate with EC100D::pir+.
The J23015 x J01064 plate grew too. Sam will grow it up on tuesday, so Jen can mate it with Ec100d::pir+.

JL_08/17/06

Yesterday, we did the Dotsanko/Wanner procedure on J23069(TraGr KO) into Rlambda x pkD46 and plated it on Cam. We also plated Rlambda x pkD46 on Cam (negative control).
The plate of J23069 in Rlambda x pkD46 grew a lawn and therefore I did a 10^6 fold dilution.
Today, Matt is doing Dotsanko/Wanner again for the OriT and trbC KO.
I am making competent cells of J23015 so I can transform J01064 into it.

JL&MF_08/15/06

Yesterday, I streaked J23055 on an Amp plate and a Cam plate to make sure that it is AmpS and CamS. Nothing grew on the Amp plate, but there were colonies on the Cam plate (which means that J23055 is CamR).
Also, I mini-preped and PCRed J23015, J23055, and pox 38, I will run a gel tomorrow.
The Rlambda x pkD3 that Matt grew up yesterday didn't grow, so I am growing it up again. Hopefully it will turn out well tomorrow, so I can do the Dotsanko/Wanner procedure for TraGr KO tomorrow.
Matt ligated and transformed pSB3C6-J23065 on the off chance that it would grow white. Hopefully the colonies will be white, meaning that the plasmid was cut correctly.
Matt also did the Dotsanko/Wanner procedure for the OriT and trbC KO. He plated them on CAM because there was no KAN + CAM available. Tomorrow he will grow them up so he can conjugate them with EC100D::pir+.

JL_08/14/06

• Jen made the PCR of PJL22 using oligos JL18 and JL19. I also grew up Rlambda x pkD46 in AmpKan.
  

Here is the picture of PJL22 (note:ladder= 1kb plus).

The band is the correct size
Tomorrow we will do the Dotsanko/Wanner procedure using the PCR of PJL22 and the Rlambda x pkD46 culture.

• I grew up a 5 ml culture of pOX38 X pKD46 so we can do Dotsanko/Wanner with the OriT and trbC KOs that Sam extracted from a gel.
• I also digested pSB1A2-J23028 with Xba1/Pst1 (large 2262) and pSB3C6- J23018 with Xba1/Pst1 (small about 700) because the pSB3C6-J23065 came out pink, suggesting that the RFP on the [TT][promoter][RBS][RFP] piece was not cut. I ran a gel and gel purified the digestions so I can ligate and transform tomorrow.
• Results from the OriT KO and trbCKO conjugation experiment with EC100D::pir+ that was done on friday:
  

1. Nothing grew on plates J23055 (TriK), J23015 (Tricam), and J23015 (TriK). This suggests that the OriT is knocked-out which is good.
2. There were >1000 colonies that grew on J23016(TriK), and J23056 (TriK), but nothing grew on J23016 (TriCAm). These are not the results that we want. Therefore, we are going to redo the trbC knockouts; we are also re-doing the Orit KO (just in case).

• Eventhough the OriT KO looks promising, we are going to make sure that OriT is actually knocked out. First, Jen streaked J23055 on an Amp plate and a Cam plate to make sure that it is AmpS and CamS. Then we grew up pox38, J23015, and J23055 so we can mini-prep, PCR, and run a gel tomorrow to see if they have the correct band size.
• Also Jen added J23067 and J23068 to the wiki. These are intermidiates to make TraGr KO. And she also added J23069 which is the TraGr Ko.

MF_08/11/06

• I sent for sequencing J23055 (Orit KO), J23056 (trbC KO), J23015 (OriT KO cam), and J23016 (trbC KO cam)
• I conjugated J23015, J23016, J23055 and J23056 with EC100D::pir+. I am plating J23015 and J23106 on TriKan and TriCam. I am plating J23055 and J23056 on TriKan.
• Sam redid the promoter + RBS for our complimentation project. We will know if that worked if: 1) The colonies are white on the plate (meaning that the parent vector was NOT ligated, and that the correct parts were ligated) and 2) After we mini prep the white colonies the sequencing comes back as what we expected.
• Jen is working on the new traGR KO (J23017). She ligated pJL20+psB1A2-Bca9007 (new name= pJL22) , transformed it, and plated it on CamAmp.
• I made pSB3C6- J23063 just in case the promoter + RBS doesn't work.

SIL_08/10/06

• Jen replated pKD46-pOX38 and they are now growing at 30 degrees and will need to be grown up tomorrow at 30 degrees as well
• She also scraped the pJL20 plate, miniprepped the colonies, PCRed them, digested, ligated and transformed the products.
• I conjugated Rlambda+pSB1A2-J01003 and Rlambda+pSB3C6-J01024 today in order to see if Rlambda could pass a plasmid into another Rlambda cell. I plated this the mating on CamAmp but also did negative controls for each.
• The conjugation results of the knockouts yesterday came back very good. All the plates are blank. Unfortunately, this wildly contradicts the results from last time where there were lawns. One possibility is that there are multiple plasmids in each cell. However, the only way to be sure is to do the experiment one more time to check to see if either of the past times was a fluke. So Matt is growing up J23015, J23016, J23055 and J23056 again to conjugate tomorrow. We will plate J23015 and J23016 on TriCam and TriKan, so Matt made more plates so that we could do so.
• Matt also made Carb plates and CamAmp plates.

SIL_08/09/06

• I miniprepped the only clone of pSB1A2-J23038 that did not turn out red (indicating the parent vector) and Matt sent it in for sequencing.
• I ran a gel of the PCRs on pKD3 and gel purified the bands, which came out very clearly. Unfortunately, there was only one MinElute column left, so I had to use a QiaQuick spin column on the OriT KOs so I eluted with 30 uL instead of just 10ul and will have to take that into account when I use it later.
• Also, made -80 stocks of J23055 and pSB1A2-J23038
• Jen regrew fresh colonies from the old pOX38xpKD46 plates, both GeneHogs and MC1061, at 30 degrees to restreak tomorrow on new plates also at 30 degrees.
• Also, we are conjugating J23015 and J23016 with Ec100D and plating it on TriCamKan. We are also mating two clones of J23055 with EC100D and plating those on TriKan only.
• The PCRs of the TraGr knockout worked today, and jen digested them with Mfel/Bglll, ligated and transformed it. She also ran a gel of the digestion to check if the enzymes were correct. The gel came out well with the correct band.

JL& SIL_08/08/06

pJL20 which was plated yesterday did not grow. I ran a gel of Rlambda digested with MfeI/BglII, but no bands showed. Therefore, I redid the PCR of Rlambda using oligos JL20&JL21.

Sam's Day:

• Conjugation assay with J23015, J23016, and J23056 came out badly with lawns for all of them. J23015, the OriT KO had slightly less but still way too more than it should have. It is unclear what is the problem except that we might have the wrong phenotype or OriT and trbC may have inserted themselves somewhere else in the genome. It is clear that the cassette is there though since they all had the CAM resistance cassette.
• Now, I did a negative control by plating J23015, J23016, and J23056 on TriK plate to see if they already have a Tri resistance.
• Also, grew up J23015 and J23016 to mate again with EC100D tomorrow and plate on TriCamKan, in order to see if the problem is that there are mixed colonies because of multiple copies of the plasmid, some of which have the knockout and some that don't.
• At the same time we are getting ready to do the knockout again just in case. I redid the PCR of pKD3 for OriTf and trbCf. I did the OriTf with a higher concentration of oligo because last time there were issues with the dilution of the original oligos. This time we are going to gel purify the band.
• Picked fresh colonies from the old pOX38xpKD46 plates, both GeneHogs and MC1061, to restreak tomorrow on new plates.
• Finally, I picked colonies from pSB1A2-J23038, the promoter plus RBS part, to miniprep and sequence tomorrow. I also grew up the J23055 clones to conjugate tomorrow with EC100D as well. I am also going to make a -80 stock of J23055 tomorrow.

Matt miniprepped the 1022 library promoters, made Tri and TriCamKan plates that will be used tomorrow, grew some overnight cultures, and helped bryan do some riboregulator stuff.

JL&SIL_08/07/06

I mapped out the PCRs (with oligos JL20&JL21) of Rlambda. Here is the picture of the gel: (lane 1= Rlambda PCR w/ H20, lane 2= Rlambda PCR w/ DMSO, lane 3= ladder):

The Rlambda PCR with DMSO showed the correct band, so I digested it (using MfeI/BglII). Also, I did the ligation using pGLN as the backbone, transformed it and plated on Amp (new name= pJL20).
The sequencing for pSB1A2-J23038 failed probably because of the bad miniprep from the promoter that i digested anyway, so sam digested the new miniprep that she made last week, ligated and transformed again. This time the digest came out very nicely so it should work this time.
Also, sam conjugated J23015, J23016 and J23056 with EC100D using the conjugation protocol involving dilutions and plated them on TriK.
She also diluted J23055 and plated it on Kan at 42 degrees.
Matt grew up cultures of all the library promoters in order to miniprep them tomorrow. He also poured lots of Kan and Cam plates because we are out.

JL_08/04/06

The mapping of OriTf KO came out well. The two PCRs of OriTf KO that we mapped used the oligos JL3& JL4, and ca850R&JL4. Here is the picture of the gel. (lane 1= OriTf KO (JL3&JL4) expected size of 1953 bp, lane 2= OriTf KO (850R&JL4) expected size of 1731, lane 3=ladder):

Sam transformed pCp20 into J23015 (poX38 OriTKO) plated it on CamAmp and is growing it at 30 degrees.
I did the PCR (fusion) of oligos JL20 & JL21 on Rlambda and RP4, but there were no bands when I mapped them. So I did a different PCR (expand) using the 4K45 program. I will map, digest,ligate and transform on Monday.

JL_08/03/06

I mapped the PCR product of OriTf KO with the oligos ca850R and OriTBB, but there were no bands. So, today I PCRed two more OriT KO's with oligos JL3 and Jl4, and with ca850R and JL4.
We got the oligos JL20, JL21, JL22, and JL23 today. These are going to be used to make a new TraG KO. We are growing up RP4 so we can PCR JL20 and JL21 on RP4 tomorrow.
Sam mini-prepped the 4 clones of pSB1A2-J23038 and the RA2 promoter.
Matt sequenced clones 1 and 2 of pSB1A2-J23038 and clones 3 and 4 of pSB1A2-J23028.
We also diluted pOX38 with a trbCKO (J23016) X pCp20 six orders of magnitude, plated on Kan, and put them in the 42degree incubator.

---

SIL_08/02/06

Today, Matt miniprepped the 4 clones of pSB1A3-J23028 which is [trbCf][TT].
Jen mapped the PCR products from yesterday of the pOX38 knockouts of OriTf and trbCf.
Here is the picture of the gel (lane 1=oriTf, lane 2= trbCf, lane3= ladder):

Only the trbCf KO came out well so I did a small scale electroporation prep on them and transformed pCp20 into them, plated on Amp and Cam and grew them up at 30 degrees. I also did a negative control for just the trbCf KO (J23016) by plating it on Amp before the transformation.
For the OriTf KO, Jen is doing another PCR with oligos 850R and OriTBB instead. And is mapping them.
File:JL080206-2.jpg
I picked colonies from the pSB1A2-J23038 part that I made yesterday which is a promoter plus a ribosome binding site. Unfortunately, Matt noticed today that the minipreps of the promoters may have been dirty because of a switch between PB buffer and PE buffer. This means that the A2 promoter miniprep that I used yesterday may have been dirty, which could explain the smeary gel. However, I cut a band from the gel anyway, so there is still a chance that of the 4 clones, one of them may be right, especially since there were white colonies and only a few red ones, which would indicate the parent vector. However, just in case, I am growing up a new saturated culture of A2 promoter to miniprep tomorrow just in case it doesn't work.
Matt ran an analytic digest and mapped the four [trbC][TT] (pSB1A3- J23028) cultures. Here is the picture of the gel:

JL_08/01/06

Today, I mini prep-ed, PCRed and mapped 10 clones of TraGr KO using the JL18&19 oligos. However, none of the clones had the correct band of 1462 bp. All of the bands were also blurry and ambiguous.
Also, Matt mini preped and PCRed the F plasmid KO of OriT and TrbC. We will map them tomorrow.
Sam made the part for J23038 [A2 promoter][RBS].

SIL&JL_07/31/06

Today, Matt miniprepped 25 promoters from the library and also made -80 stocks of ten of them. He also sent out for sequencing promoters A-F and four randomly chosen promotoers throughout the strength spectrum.
I miniprepped the TraG and TraM complements with double terminators pSB1AKG0-J23026 and pSB1AKG0-J23027. Then I digested them in EcoR/Pst and mapped them to check if they are the product desired (wouldhave 2 bands at 3151 bp and 2078bp (for J23026) and 421bp (for J3027)) or if they are the parent vector (would have 3 bands at 3151 bp, 1459 bp and 844 bp).
Picture of gel from EcoR1/Pst1 digest (lane 1 = pSB1AKG0-J23026 (band1) clone 1, lane 2= pSB1AKG0-J23026 (band1) clone2, lane 3 = pSB1AKG0-J23026 (band2) clone 1, lane 4 = pSB1AKG0-J23026 (band2) clone2, lane 5 = pSB1AKG0-J23027 clone 1, lane 6 = pSB1AKG0-J23027 clone2, lane 7 = ladder)

lane 4 is most likely the correct pSB1AKGO-J23036. lanes 5 and 6 came out pretty ambiguous which i think is the fault of the minipreps, so I miniprepped pSB1AKGO-J23027 clones 1 and 2 again and remapped them.
Picture of pSB1AKG0-J23027 digestion (lane 1= clone 1, lane 2= clone 2, lane 3=ladder)
File:SIL073106-2.jpg
The gel came out negative again for pSB1AKG0-J23027 because there is only one big band, which is unfortunate. However, the TraM complement with double terminator is not that critical because we are not really following the TraM path anymore so we are just going to forget about it.
I am also growing B0034 from -80 in order to miniprep it tomorrow and make a part with a promoter (A2) and RBS tomorrow.
I also added the double terminator to the trbC complement by ligation and transformation.
Also, Jen PCRed the oligos of the TraG KO 20mers and is growing up saturated cultures of the pOX38 knockouts of trbCf and OriTf.
Picture of gel of TraG KO 20mers (lane 1= TraG KO clone #7, lane 2= TraG KO clone #8, lane 3= ladder)

Both clones #7 and #8 have bands that are around 2000 bp which means that they are both the wild type TraGr (wt TraGr is expected to be 2246 bp long, but TraGr KO is expected to be only 1462 bp long). Therefore, today we picked more colonies from the TraGr KO plate and grew them up, and tomorrow we are going to mini prep and map them. Hopefully, we will find a colony that will have the TraGr KO. Also, Chris is going to design a different TraGr KO cassette in case all the colonies we map are wt TraGr.

SIL_07/27/06

Today, Matt did the knockouts of OriT and trbCf on the F plasmid (both poX38xpKD46 variants GeneHog and MC1061). He also did some library stuff.
Jenn minipreped, mapped, and sequenced the trbCf complement pSB1A2-J23014.
Here is the gel from the trbCf complement analytic digest (lane 1 = ladder, lane 2 = clone 1, lane 3=clone2)

I am growing up overnights of pSB1AKG0-J23026 and pSB1AKG0-J23027 the TraG and TraM complements with double terminators so that they can be miniprepped and sequenced. Unfortunately, when I was making pSB1AKG0-J23026 the digest resulted in more bands that expected, but I cut out ones that seemed to be at appropriate lenghts and thus we have 2 variants of the plasmid which need to be sequenced in order to determine which one is the correct one.
Also finally, I transformed the helper plasmid pCP20 into J23017 (Rlambda TraG KO) and plated it on Amp Cam at 30 degrees. I had to do a small scale electroporation prep to make the competent cells in order to do this.

MF&JL_07/26/06

We picked colonies, one from each of the plates, of the four plates that we plated yesterday from the conjugations( pOX38 w/ pKD46 in Gene Hogs conjugated at 30 degrees, pOX38 w/ pKD46 in MC1061 conjugated at 30 degrees, pOX38 w/ pKD46 in Gene Hogs conjugated at 37 degrees and pOX38 w/ pKD46 in MC1061 conjugated at 37 degrees).
Sam grew up colonies of trbC compliments so she can mini prep them on Thursday. She also grew up the Rlambda w/ [TraG] KO (JO23017) from the plate that we streaked yesterday.
Picture of the map of clones 7A, 7B, 8A, and 8B. (in that order)

I did the PCR and maping of clones 7A, 7B, 8A, and 8B again. The second time, no bands were seen.

MF&SIL&JL_07/25/06

We have conjugated the pOX38 with some of Chris's stocks of pKD46 (in GeneHogs and MC1061) . We will have to see tomorrow if his stocks were still healthy.
Also, the trbCf complement PCR worked today (affirmed by a gel) and we have cleaned up the PCR, digested it in dPN1, EcoRI, and SpeI, then cleaned up the digestion, ligated with pSB1A2-I13511 digest in ecoRI and SpeI, and then transformed it into TG1 cells.
The sequencing data came back for the TraM and TraG complements and they are positive for both, so they are tough tagged and put in the freezer.
Finally, we are doing genomic minipreps of the RlambdaTraGknockout clones, from here on known as J23017. We then PCRed them with JL10 and JL11 oligos and hopefully when we map them, they'll show a band around 1200, instead of 2000 like last time, which indicated wild type Rlambda.
Here is the Rlambda traG KO PCR's Clones 6,7,8:

Clones 7 and 8 seem to have the correct band of the TraGr knockout, but they also seem to have the wt TraGr. Therefore, we restreaked clone 8 onto a Cm plate to try to seperate the wildtype from the knockout. Also, we did four more PCR preps (fusion) for clones 7 and 8. This time we used oligos JL10 and 1016R (ie. 7A, 8A), and we used oligos 824 and JL11 (ie. 7B, 8B). We did this in order to make sure we have the TraGr knockout.
We are also adding double terminators from pSB1AKG0-B0015 digested in Eco/Xba to the TraM and TraG biobricks by digesting them in Eco and SpeI.

MF_07/24/06

Today we started cultures of pOX38 and pKD46 (in GeneHogs and MC1061). The pKD46 was from Chris's -80 stock. We are redoing the mating and the knockout because pOX38 mated with pKD46 aquired Cloramphenicol sensitivity somewhere in our procedure. Also, our four plates that we plated on Friday gave us unexpected results. All four plates came out with yellow colonies and smell like their is some kind of bacteria. We believe that DH10B is the culprit in this instance. We think that DH10B might have been contaminated before we began our experiment.
However, we are proceeding with the Rlambda TraG knockout by doing a genomic miniprep of the 4 cultures we grew up Friday, then PCRing with the TraG complement oligos JL10 and JL11, and finally mapping them to see if the size of the PCR fragment can give us a conclusion on whether TraG has been knocked out.
Also, the parts registry has been updated with all the complements and knockouts.
We are still waiting for out the sequencing data for the TraM and TraG complements.
We got our trbCf 20mers! So we are PCRing them with Olambda as the DNA template.
Mapping of cultures that we started on Friday was not successful. Gel electrophoresis showed three failures and one WT traGr. So, we are growing eight more over night cultures of J23017 [traGr] from Rlambda.