Berk2006-Sequences

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Sequence files in ApE format

download ApE


Image:PAC997.str
Image:PSB1A2.str
Image:PSB1A3.str
Image:PSB1AK3.str

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#F54D70 #5DFC0A #48D1CC #FF5333 #CC00FF #EEC900

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072506

072506

I’ve decided to keep just one notebook from here on. It gets too confusing to figure out what experiments/constructions are in which file. It would be easier just to have it all in one place and be searchable. I think what I’ll do is keep separate word files with all the embedded bits and pieces and then put just text on the wiki page. I’m repeating the last few days here for convenience sake.

 

Saturday, July 08, 2006

I mini’d and worked up for sequencing Bca9004, Bca9009, Bca9018, and Bca9021. At one point I had a mixup of 9009 calling it 9003. For some reason I had labeled the plate 9003. I think I fixed it all—the -80 stock and mini are labeled 9009. Anywho, we’ll send that out Monday.

 

I did a bunch of subcloning today. The oligos to do TriR and CmR cassettes came, so the 4 parts involving those were in the mix. Total I did 11 subclones:

There was some gel purification and analytical PCRs. They were assigned letter codes according to:

The gel goes: 1015 pcr; 1016 pcr; b0015 pcr; mw; b0015 pcr; A; f; F; e; ; d; d; d; d:

Sizes all look reasonable, but I didn’t check all that carefully. The band pattern of the “d” digest of Bca9008 (BglII/MfeI) confirms the presence of the restriction sites. I don’t plan to sequence 9008 directly, I’ll get a de factor read from parts put in. I put in wbbL, invasin, and 2 rfbC hits today. So, these cassettes are RBS-ORF composites, no terminators. Regardless of the sequence of J01008, I’m going with this clone. If there are points, I’ll change the model sequence. The last 2 parts are part-building parts. 9028 is like 1008, but it includes a dblTerm downstream of the BglII/MfeI insertion sites. So, the product of conversion is an RBS-ORF-TT composite. Part 9027 is for conversion of biobricks into pBAC874t via a NotI/EcoRI subclone. It destroys the NotI site between EcoRI and XbaI so that the one between SpeI and PstI is unique. It also puts in dblTerm. To put parts into pBca9027, I would subclone into SpeI/AlwNI, then transfer NotI/EcoRI into the BAC. I’ll be using that guy for the O-antigen AND gate.

 

Wednesday, July 12, 2006

More subcloning fun. The 4 subclones into pBca1008 didn’t go so well:

They all map funky, so I’m redoing all that stuff with proper cut and paste with gel purification. I got good clones of all the constructs from the other day except Bca1015. I’m just redoing that guy. All the CmR-related subclones look fabulous.

I’m not going to sequence the CmR and TriR basic parts at all, I’m going for de facto sequencing at a later point. I want to proceed all the way to the [FRT][*R][FRT] parts asap to test FRT function. I also got in the PspOMI enzyme, so I could do the Bca1007 and Bca1009 digests. Both digested fine, so they are ok. So, I proceeded with the following subclones:

 

….and set up the digests as:

 

The gel below goes AaBbCcDG, all are good. This gel confirms the general size of the CmR-FRT, CmR, and TriR-FRT parts.

The gels on d,g,h,i,j etc. initially didn’t go so well. I was able to get only d,i, and j out of that gel, the rest looked like shit. I didn’t even save the gel. Originally, I was doing a different set of subclones, so that’s not here, it included mgrB. For the remaining ones on the list above, I went with inserts either from PCR (flu,fdhf, wbb992) or existing digests (AraInv). So the first list above is the “correct” list. I’ll need to make new maps of some of this—I don’t have a map for flu or fdhF, and wbb992 will replace the TetWbb-derived subclone. I just relegated the 1015 basic TriR part, and plated on both Amp and Tri/Amp to make sure I got cfus in case there is something weird (like TetR colony reduction) with Trimethoprim resistance.

 

I’ve sent a bunch of clones from the last batch for sequencing.

 

Wednesday, July 12, 2006

I got sequencing back from the stuff I sent, most look good:

 

There was an error in the model for Bca9028. I had part b0015 in pSB1A2, and in reality it is in pSB1AK3 (and the registry says so). When I remade the model file with the correct starting sequence, the sequencing matched the new model for what I’m going to call pSB1A0-Bca9028. All good, no biggie. It is not a pSB1AK3 as the KanR marker would not be transferred by the sub method (I don’t think…maybe check it if it becomes an issue). Part pSB1A2-Bca9009 matches the model, but the read didn’t make it past the part junction. I’ll need to do confirmation by PCR rather than sequencing. pBca9027 is not correct, I need to examine more clones. Unfortunately, this list doesn’t get me to more subclones, so tomorrow will just be characterization of all sorts of stuff. Mainly, I need to confirm the FRT-*-FRT constructs and make sure that pCP20 excises the fragment properly.

 

Thursday, July 13, 2006

I threw out the sequenced clone of Bca9027 and grew up 4 more. I did characterization of various things:

The gels (PCRs 1,2,3,Mw,a,b,c,d,e,f,g,h) are:

The Bca9009 is confirmed (combination of sequencing and PCR “1”). The first half of 9010 is confirmed, I think I’ll do some mapping for the Tnp half. The PCR failed, but it is probably right—just too long for a 2K pcr. At least I hope so….we’ll find out tomorrow. The results of mapping go:

Bca9007

[FRT][CmR][FRT]

consistent

Bca9015

[Psal]

consistent

Bca9026

[Flu]

consistent

Bca9014

[wbbL1]

consistent

Bca9013

[inv1]

off

Bca9031

[P_sal-rfbC1]

consistent

Bca9032

[P_sal-rfbC2]

consistent

Bca9033

[fdhF]

consistent

So, all but invasin are ok.

 

Saturday, July 15, 2006

I got some sequencing results back:

All are fine, didn’t get the full rbs for the wbbL rbs, but got some of it looking at the trace, and it’s fine. Going with it.

 

I’ve done some EcoRI/PstI mapping of more clones of the ones that were problematic:

Bca9005-still wrong

Bca1015-still wrong

Bca9027-1,3,4-good

Bca9027-2, wrong

Bca9013-1,2-good

 

I’ll stock and sequence 9027-1 and 9013-1. Holding onto the minis of the other 9027’s till get sequence confirmation.

 

Wednesday, July 19, 2006

 

The new#1 clones of pBca9027 and pSB1A2-Bca9013 are fine. The region downstream of PstI in 9027 still isn’t matching template, but it won’t affect the usability of the plasmid, so no worries.

 

I did some mapping of recent subclones:

They go:

1                    pSB2K3-Bca9007-1

2                    pSB2K3-Bca9007-2

3                    pSB2K3-Bca9007-3

4                    pJ23006-Bca9034

5                    pSB1A2-Bca9005-1

6                    pSB1A2-Bca9005-2

7                    pSB1A2-Bca9010-1

8                    pSB1A2-Bca9010-2

 

The mapping below is all EcoRI/PstI. Clones 1 and 3 of pSB2K3-Bca9007 were bigger, healthier clones. They appear to be the cotransformed guys, though. Clone 2 was sickly but maps right. The rest of these guys all map correct. I did 1015 (TriR alone) in this batch. It again didn’t give any triR colonies. Whatever. I’m done with that for now I think. Samantha made a SpecR basic part which is probably more user friendly. Bca9010 went very smoothly this time, many CmR/AmpR colonies, and they mapped right. Will need to PCR map those, though to confirm the Tnp presence.

 

Friday, July 21, 2006

I got sequencing results for pSB1A2-Bca9005 (F-Tri-F). It was perfect. I have still been unable to make a TriR basic part. Very weird there, since the composite works great. BTW, this is also defacto sequencing of Bca9002 since the read got the entire cassette. All good, both FRT’s present. I also got sequence on pJ23006-Bca9034, and it too is perfect.

jca293 072006 pSB1A2-Bca9005 1 ca998 perfect

jca294 072006 pJ23006-Bca9034 1 ca998 perfect

 

 

To confirm pSB1A2-Bca9010 (CmR.Tnp), I did PCR w/ 998 and 9014R, expected size 2721 bp. The third lane on the gel below is that. Looks solid.

I wanted to confirm the activity of FRT (if I could) at this stage, so I moved the Bca9007 cassette into pSB2K3 and then transformed pCP20 comp with that guy. If the thing flps, the EcoRI/PstI region changes size. The expected fragments would be:

No flp pSB2K3-Bca9007 3446+1957

FRT deletion 3446+1013

The bands in the middle are a miniprep of a clone grown ON in just kan (to clear pCP20, maintain the pSB2K3). The result is a little fishy. The digest btw is XhoI/PstI in neb2. It looks like the 3446 band is there, and the plasmid is a partial digest. The fragment band that would correspond to the F-Cm-F fragment is too big. It’s either a map error, or some non-expected recombination occurring. I’m not convinced this is a valid experiment—this might not fly to do it this way. The small band around 600 bp isn’t an expected fragment either. Not sure how that comes to be.

 

The first band on the gel comes from transforming pKD908b/pir116 with Bca9010. Those cells did not look so healthy on the plate, kindov small. I grew one up in just Amp, and it looks like the cells just kicked out the R6K plasmid. BTW, that’s a SpeI digest which would linearize all relavent plasmids. So, that’s no confirmation of Tnp function. I would really like to see some evidence of FRT and Tnp function before proceeding, but I’m not sure how else to assay it. I’m only 2 steps away from being able to test the Tnp final construct. So, I’ll just test at the end and hope for the best. I think I’ll also just have to wing the FRT stuff until there is a genome-integrated cassette.

 

I miniprepped and -80’d clones of the following constructs:

I’ll do de facto characterization of those since they each manifest a phenotype.

 

I did a series of subclones today…

Hh and Ff insert S03155 looked like crap, so I grew up more to miniprep and redigest. The large fragments for each were fine.

 

Monday, July 24, 2006

pBca1020-bca9015 perfect

pBca1020-Bca9022-1 perfect

pBca1020-Bca9022-2 wrong

pBca1020-Bca9022-3 wrong

pSB1A2-Bca9006 wrong

pSB1A2-Bca9008 perfect

pBca1020-Bca9036-1 perfect

pBca1020-Bca9036-2 perfect

pSB1AK3-Bca9037 perfect

pSB1AK3-Bca9038 extra band (contam?)

 

Keeping and -80’ing the ones in red. Growing up more 9006 and 9038.

 

Tuesday, July 25, 2006

I mini’d and mapped some more constructs. I don’t know what’s up with the trimethoprim marker, but I’m starting to think I should just ditch it. Yeah, I think I’m going to do that and maybe replace its use with the specR parts. This just isn’t happening.

pSB1A2-Bca9001 perfect

pSB1A2-Bca9006-1 weird

pSB1A2-Bca9006-2 weird

pSB1A2-Bca9006-3 weird

pSB1A2-Bca9006-4 weird

pSB1A2-Bca9029 weird

pSB1AK3-Bca9038-1 right, I think

pSB1AK3-Bca9038-2 wrong, I think

 

The pBca1020 series plasmids is working out just fine now. Initially I tried putting the RFP into the Salicylate promoter part (9015). I got no red colonies, so I put it into R0040 which gave the expected phenotype. I took one of those guys and subcloned in the IPTG(9022) part and the Sal(9015). The sal ones came out white w/o sal, the IPTG ones were red without sal. I grew up the Sal clone with 100 ug/mL sal, and the cell pellet was pinky, but way lower expression than with tet promoter. A little weird. I was worried that the IPTG part wasn’t inducing, so I tested the 1020 with an induction series—it’s just very leaky at high copy, but that should settle down at single copy I think (below). The half-max seems to be around 5 ug/mL of IPTG. Good enough, going to proceed with putting on wbbL.

 

I subcloned the extension product for the 1022 library into the sal construct (pBca1020-Bca9015) and got ~5% pinkish colonies. Matt grew up 96 pinks, 96 whites, and I reastreaked 16 too-dense clones that were very red. We’ll sort through that with the tecan tomorrow.

 

I did several new subclones today:

 

Wednesday, July 26, 2006

I did PCR to confirm the composition of Bca9001. They are:

 

KB001/ca641F TT 2048bp

KB009/ca641F Ptet 1884bp

Looks solid, proceeding with the subclones to do neuD and wbbL knockins:

 

Yesterdays subclones all gave tons of colonies, growing up one of each.

The 1022 library gave the following profile. This is FluorRFP/OD600. The whiter ones all were from white colonies, redder from red, so that’s consistant. The parent construct (Psal) comes out around 55 on this chart. Ptet is at the far right, comparable in activity to the best 3 hits. I restreaked multiple colonies as well, grew up some of those to assay tomorrow. Matt grew about 18 clones across the spectrum of activity to assay tomorrow by Tecan/Cytometry and then mini, stock, seq.

 

Thursday, July 27, 2006

 

pSB1A2-Bca9023 wrong

pBca9020-Bca9030 good

pSB1A2-Bca9020 good

pSB1A2-Bca9011 good

 

Saturday, July 29, 2006

The pBca9020-Bca9030 (2 clones, only clone 1 mapped and stocked) both gave a light pink pellet upon pelleting an LB w/o Mg culture. #1 mapped right, so I don’t think I’ll characterize further. The sal and Mg NoB subs both came out as weakened promoters, which is weird. It’s almost as though the restriction sites pinned between the promoter and the ORF are terminators. I might have to redesign that. It definitely means I don’t want to do the Sal-RfbC splitting them up.

 

I did some mini and mapping:

pBca9012-1 large frag too small

pBca9012-2 large frag too small

pBca9012-3 large frag too small

pBca9012-4 correct (dominant phenotype, light pink)

pGLN-Bca1004 good

pSB1A2-Bca9035 good

pSB1A2-Bca9023-1 wrong

pSB1A2-Bca9023-2 wrong

pSB1A2-Bca9023-3 wrong

 

The scheme for construction of pBca9012 was:

That went very smoothly, got tons of small colonies in Ec100D::pir+. At first they all looked white and I was worried. A few were very red, and I grew clones 1-3 from those. Clone 4 was a representative white colony. Only clone 4 maps right, and it was pink after saturation. The plate colonies have all turned pink on the bench after a few days.

 

I did midipreps of pSB1A2-J23012 (SpR), pSB1A2-Bca1010 (FRT), pSB1A2-Bca9008 (F-Cm-F), pBca1020-Bca1022-C2 (Pcon). I’m digesting 23012 and 1010 and 9008 with XbaI/PstI/CIP. I would do it with AlwNI instead of PstI, but we’re out. I’m also doing 9008 with HindIII/SpeI/CIP. Those are all being gel purified. I purchased oligo CA1024F (biotinylated ca998) and did PCR ca1024F/G00101 on pSB1A2-Bca1011 mini with Taq on 200 uL scale and gel purified all of it.

 

I’m going to try solid-phase biobrick assembly with the above digests and some BioMag (Bangs labs) nuclease-free streptavidin beads. These things are 4.4 ug/mg biotin at 1.2 mg/mL for a binding capacity of 22 uM for biotin. So, 25 uL of beads has orders of magnitude more capacity than does 100 ng of a 500 bp DNA fragment capped in biotin. That’s the volume I’ll start with for assembly.

 

Since all the added cassettes are CIP treated, the oly 5’ phosphates are on the bead. This should address the stoichiometry issues. The last step will be to ligate on the SpeI-9008-HindIII plasmid cap, digest off the resin with EcoRI and ligate to circularize. I might then hit half of it with T7 or T4 pol to fix the nicked strands, then transform. Some of the midipreps lost the pellet—I did this by the standard HiSpeed method which was stupid. So, of the things I have to start with the best two constructs to try are a Spec-FRT part and a Spec-FRT-Spec part. I think I’ll try both…we’ll see how I feel about that on Monday. The little nicks due to CIP might become an issue with multiple parts. Not sure. All the nicks will be on one side of the DNA, so the thing can’t fall apart, but it might not transform well.

 

I don’t know what’s up with 9023—I rechecked the sub scheme, and this guy really should be clean. I think I’ll just redo it Monday—I must have just made an error somewhere. I should probably start taking pics of the subcloning gels to be sure so I can look back at the source gels and know if anything was weird. The colonies are white, and they map as the wbbL plasmid. That’s the small frag, so it really should not have bled anything through. Hmmm.

 

Monday, July 31, 2006

I’m doing the digests for the solid phase assembly:

All are looking good on the gel thus far. Doing a long gel to resolve the 1184/929 one. Red is the desired band for each.

 

Meanwhile, I’ve started the beads. I measured the amt of DNA in the biotinylated guy at around 75 ng/uL. I’m doing 2 uL scale, but 2 of them, so I put 4 uL with 50 uL of beads. The beads were washed 1x in 700 uL PBS, resuspended in 500 uL PBS, added the DNA, incubated 30 min, washed with water 1x, brought back up in 200 uL of 1X NEB2 + 1 uL XbaI. Digesting about 1 hr.

 

I did PCR mapping on 9020 and 9012:

The pSB1A2-Bca9020 PCRs are perfect. It confirms the location and presence of R6K, CmR, and OriTr. It turns out I don’t have probes for FRT or TnRev, but the part junction is confirmed here. I am proceeding with the subclones.

 

On the pBca9012, the presence of CmR, Tnp, OriT, and R6K is confirmed…the sizes are all crazy. I interpret this as being that the frags were too long for the ext time and I get a mispriming with 1002R. The “C” band has the correct size and the mispriming size, but the rest are all mispriming, but they appear to all misprime at the same two general regions. I’m calling that good enough, will focus on getting a function confirmation here.

 

The status of the 1022 library is as follows. Matt re-picked the following clones from the screen of 192 clones:

C2, D7, C5, H12, E1, B10, E11, F3, A5, F6, E10, C1, B9, F4, D7, C2, F12, A2, E8

I grew up 6 clones (AàF) of the ones that were “very red”. Indeed they were very red at saturation, some more than Ptet. The large set of clones that Matt picked were well-isolated, so I am not restreaking those. I did -80’s on those over the wknd, Matt is miniprepping today. The AàF guys were clearly mixed, so I restreaked those from the culture, picked more cfu’s yesterday. Matt -80’d and mini’d those today. All the Tecan data is in the file “072606-Tecan data…”:

The data was mostly consistant with the first run, only a few changed position. The values on the AàFs is from the mixed culture, so those aren’t valid numbers. I think the next step is to do some sequencing and see what we’re dealing with in terms of miniprep quality, degree of cotransformed hits, and sequence diversity. Clearly I have a nice broad range of hits here.

 

Doing some subcloning today:

The 9023 is a re-do from before. Hopefully it will come out better this time.

 

PCR with the 1023 20mers of irp9 with Phusion still looks like shit:

The right lane is a 1019 pcr off the 1023 pcr. Not real sure where to go next there. I think it’s some TA kit thing, though. I think I’ll wait until my new Expand kit comes in. Phusion seems to generate a product even if there isn’t an appropriate template. It primes too well.

 

The PCRs of the knockout with Phusion also looked shitty:

Again, I think I’ll wait on the Expand kit. The M band is the wrong size. I’ll recheck that 279 is the correct oligo to do neuD.

 

The Bca1025 AGGA mutant of pBca1021-E0040 (pBca1020-Bca1025) came out as about 100 cfu, all green. I will redo that guy by overlap:

 

Having some difficulty on the solid phase assembly. The digests all looked flawless. The beads, however, appear to be sticking and/or degrading. They don’t seem to be stable to multiple rounds of incubation in buffer. Not sure how to remedy that.

 

I remapped the pSB1AK3-Bca9038’s with BamHI/PstI and got the gel below. Clone 1 is good, 2 is a dud.

 

Tuesday, August 01, 2006

I did the solid phase assembly again today to make a FRT-Spec-FRT part in pSB1A2. I first loaded 25uL of beads directly onto the pad of a minelute column. The liquid immediate went into the column, so I added 200 uL of water and pipetted up and down to distribute it a little. I added 100uL of 1X neb2, 1 uL XbaI, and 2.5 uL of the biotinylated PCR product. Incubated 0.5 hour then did 100uL scale digestions and ligations with 1uL SpeI or 1uL ligase + 4uL biobrick part. Did:

Eluted the qiaprep in 10uL, setup ligation normally, transformed all of it, plated on Amp and Amp/Spec. We’ll see what happens!

 

The Bca1025 overlap PCR looked really good. I sub’d EcoRI/SpeI, went directly into the pSB3C6 variation. I won’t be able to confirm activity, but hopefully the tRNA that Kaitlin is making will all go well. I might move the entire tRNA cassette into something just in case.

 

Bca9023, Bca9016, and Bca9017 all gave oodles of colonies, grew up 2 of each to screen tomorrow.

 

I setup new PCRs of pGLN-Bca1004 and pSB1A2-Bca9035 to do the neuD and wbbL knockins. Grew up Bos12/pKD46 from the -80 to do the comp.

 

Wednesday, August 02, 2006

The bands go:

so, looking good. Stocking and saving the ones in red. I gp’d the bands, doing the following subclones:

The subs all went smoothly, transformed TG1 or pir116 heat shock cells.

 

The F-Sp-F plates came out promising. Got about 200 cfu on Amp, about 15 on Amp/Spec. I grew up 16 from amp and 8 from Asp to screen by colony pcr tomorrow.

 

The neuD and wbbL knockin pcrs came out good with expand, so I dig’d with DpnI and transformed Bos12/pKD46. Hopefully we’ll have clones tomorrow!

 

The pSB3C6-Bca1025 plate gave lots of cfus, none were green, grew up 2 to screen.

 

Thursday, August 03, 2006

I got no colonies for the bos12 knockouts. Will repeat tomorrow at higher OD.

 

I mini’d and passed off the Bca1025’s to Kaitlin for her to stock and sequence. I’ll put the construction file up tonight.

 

The 9039 and NES subs looked great. Light pink for pBACr-NES, grew up 1. Grew 2 of the pSB1AK3-Bca9039’s. Tomorrow I will put the wbbL and rfbC cassettes into pBACr-NES and also blunt out the XbaI site for pBACr-NESd.

 

Adam Deutschbauer’s dap variant of BW20767 (WM3064 I think) grew fine on the plate, grew up a colony to do comp tomorrow.

 

The pBca9045 and pBca9046 gave shitloads of small colonies. I grew 2 of each. I probably should have done a neg on the cells, it looked a little sketchy, but hopefully correct. Those are in pir116, so they might be sickly.

 

The solid phase assembly of FRT-Spec-FRT looks funny.

The gel is of 24 clones as Taq G00101/ca998 colony PCR. None are obviously correct. The markers are the 1kb, I’d expect something like 1.1kb. The 16 on the left were naïve, the 8 on the right were specR. I don’t really need the construct, so I think I’ll just move on to a new strategy—doing it on plates like an elisa.

 

Still having trouble cloning salS. Not sure I really want to put more effort into this guy. I did 1023F/R pcr with Expand on Y pseud. gen. Got multiple bright bands. 1019F/R pcr on that gave no band. I setup 1019F/1023R and 1023F/1019R to see if it’s just one of the 1019’s that fails. If so, I’ll just TA it and go from there.

 

I also sub’d the 901F/R mgrB pcr into pBAC583 (old NoB dig). Trans TG1. The purpose of this is to revisit the pir116/R6K regulation library.

 

Friday, August 04, 2006

I did some mapping: first lane is pBACr-NES, second two are pSB1AK3-Bca9039, all are EcoRI/SpeI digs.

Looks good, going with 9039 clone 1. I made -80’s of those and WM3064, then subcloned 9037,9038, and 9039 with ES into pBACr-NES.

 

The 9045 and 9046 guys are clearly massively contaminated. The lawn is that funny yellow junk. There are a few pink guys in there, I picked one from each plate and restreaked it today. Will grow them up tomorrow, hopefully. For the BAC transformations I’m using the same cells but doing Kan/Tri, and hopefully that will kill the garbage. I’m also running negs on K and C for the cells to see what’s up.

 

There were some colonies on the neuD/wbbL knockins, so I just put the bos12/pKD46 in the fridge and grew up the colonies.

 

pBAC901 looked solid. ~0.5% strong green cfus, rest were light green. Picked a light green. Will mini and sub the pir116/R6K lib tomorrow (if I can find it).

 

The SalS PCRs failed again. I think I’ll just drop that…not worth the trouble.

 

I did a standard Vent blunting of pBACr-NES to make pBACr-NESd. This guy has everything biobrick-unique except PstI.

 

Monday, August 07, 2006

I grew up and gen prepped the one colony of the neuD knockin (L1) and two of the wbbL knockins (M1 and M2). I did PCRs and EcoRI digestion to map:

The two wbbL knockins look good, neuD looks like it may be a dud…maybe.

 

Did some mini and mapping:

 

pBca9045

pBca9046

Bca9037-1

Bca9037-2

Bca9038-1

Bca9038-2

Bca9039-1

Bca9039-2

 

Thursday, August 10, 2006

I mini’d and mapped 2 of 9040, 9041, and 9042, the pBACr- Piptg-wbbL and Psal-rfbC’s. Looks a little weird (XbaI digs):

It’s all too big. I think I need some PCR mapping to see if they are in the ballpark or way off.

 

The mgrB/pir-r6K business gave a lawn of bacteria. I scraped the plate, mini’d, did 901F/875R PCR and got out a 1kb band—too small. I have concerns about pBAC901—it wasn’t green enough in the pellet and there were some faint additional bands in the digest. Something’s up with that guy.

 

Tuesday, August 15, 2006

I did some PCR mapping of the pBACr-Bca9040, Bca9041, and Bca9042 with ca605F/ca606R. I got no product but did using pBACr-NES as a control and it gave a good product. So, I think the pir116 is still having contamination issues. The mapping of the pBca9045 and pBca9046 also came out weird, I think it is all just contamination in that strain.

 

Today I re-ligated pBca9045 and pBca9046 and transformed directly into BW20767. Hopefully that will work better—these are the cells the kids made, and they transformed pBca9012 pretty cleanly (almost all red colonies, I grew up one did a -80 today).

 

I did a small scale comp of pSB3C6-Bca1025 (the GFP-AGGA guy) and transformed in Kaitlin’s two tRNA constructs. I got tons of cfus, but I didn’t do a neg control. Suffice it to say, they weren’t green on the plate or upon growing to sat’n. I’m not sure if this is just a contaminated prep (seems unlikely) or no activity in either the reporter or the tRNA. My plan is to move the Bca1025 reporter back up to pSB1A2 and put it with the original Ser2-AGGA reporter.

 

I ordered a new oligo for doing pir116:

ca1026R Reverse oligo for pir116

CCATAgaattcGCCATATATCACCCCTTAGC

 

The pcr went fine, ligated into pBACr899 and pBACr-mgr901. I got no colonies on SalK for the 899 variants and all green colonies on the 901 plate. Not sure why that’s going wrong. I’m suspecting that either there’s a toxicity thing or the BamHI site on the 977F oligo is screwed up. I’m going to take an alternate route here:

 

PCR ca56/ca1026R on pBAC905 (bp, BamHI/EcoRI)

Sub into pBACr-Mgr901 (BamHI/EcoRI)

Product is pBACr-MgrPir56

EIPCR ca901R/ca977F on MgrPir56 (bp, BamHI/Vent)

Product is pBACr-MgrPir977 lib

 

That should take care of any restriction site issues.

 

Thursday, August 17, 2006

All the subs yesterday went well…

pBACr-MgrPir56 (described above, got about 100 cfu, grew up 3, in TG1)

Bca1025 into pSB1AK3(-b0015) (about 500 cfu in TG1, grew up 2), it did this because the tRNAs weren’t green. Going to put this new reporter with the old pAC-Ser2AGGA to test the reporter.

pBACr-90* (40,41,42) resub’d into pBACr-NESd the original scheme, transformed into BW20767, got ~50 cfu of each, looked much more normal.

 

I religated the pBca9045/9046 the other day, transformed directly into BW20767. The 9046’s are decently red, possibly full on. Cell pellets of 9045 are pinkish, colonies are white. I did triparental mating with two individual clones of these guys with pBca9012/BW and RU1012 as the recipient, plating on KC. I got dusty lawns, looks like transposition, though, for the ones where 9045 or 9046 was added. I should have added aTc to these, but forgot. It all seems to work, though. I’ve replated MC600u, going to transform pBACr-UG784 (Ptet-uppgen), show first that those are genR,uppS, then do transposition and see if I can find some interesting promoters with that.

 

I’m getting some P1vir from the Bustamante lab tomorrow, the protocol for transduction is here:

http://www.openwetware.org/wiki/Sauer:P1vir_phage_transduction

Going to try that on the knockins of Bos12 for wbbL and neuD. Going to both try to transfer the cassettes back to Bos12 and pass them to MG1655 and MC1061. It would be really nice if that all could work in MC1061. We’ll see.

 

Friday, August 18, 2006

The above gel(s) goes:

ES pBACr-9040-1

ES pBACr-9040-2

ES pBACr-9041-1

ES pBACr-9041-2

ES pBACr-9042-1

ES pBACr-9042-2

ES pSB1AK3-Bca1025-1

ES pSB1AK3-Bca1025-2

BE pBACr-MgrPir56-1

BE pBACr-AraT7940-F11 from pir116 for voigt stock

 

So, all look good except pSB1AK3-Bca1025-2. Did -80s of all the clone 1’s and saved minis. I transformed MG1655 or MG/pMF19 with the relavent pBACr904*’s. I’m going to do the P1 transduction business on several guys and do all the results together on one O-gel.

 

On the neuD/wbbL knockins, I had another colony on the “L” plate (the neuD knockin). I grew that up yesterday. It gen-prepped like an E. coli, and I setup the analytical PCR:

We’ll see how that looks tomorrow. I did an amplification of the Bustamante P1 stock. It totally cleared my culture of MC600u. So, that’s good. I’ll do the transduction tomorrow and try sending things into MG1655, Bos12, and MC600u. So, I grew up cultures of all that today.

 

I also transformed MC600u with pBACr-UG784. I’ll do the first transposon library on that.

 

Since the pBACr-MgrPir56 looks pretty good, the mini clearly shows higher copy even though it’s in TG1, so it must be working to some degree. I setup a PCR:

That will give a plasmid that looks like:

If I do the closure of the circle with NotI, the site will be destroyed. I can also close it up with BglII partial digestion. I haven’t decided yet which is better. The gameplan is to do this, make sure the thing replicates, then make the EIPCR library directly on it to make the Mg-sensitive replicon (if this guy isn’t already Mg-sensitive).

 

The riboregulator stuff is looking really good, so I don’t think we need 4-base anymore. I have the Bca1025 in case we need to revisit it. If I revisit all that, this guy needs sequencing (it was a sloppy subclone) and then can put in pAC-Ser2AGGA to confirm activity. If that works, can revisit the biobricked four-base.

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