Archive for the ‘Experiments & Data’ Category

Naked MudJ Revealed

Monday, November 8th, 2010

Using Solexa methodology, I recently sequenced strain SMP1666 (from Sophie Maisnier-Patin), containing 3 copies of MudJ. One had lost the infamous trpB-proximal stem-loop structure1,2, and the other two were pristine — one hesitates to say “wild type” as the lollypop structure was itself an artifact of the original construction out of a MudI background3. The complete MudJ sequence can be found here. The big surprise is the cynX & cynS genes between lac and the Tn5 drug-resistance cassette.

1.J.Zieg & R.Kolter (1989) “The right end of MudI(Ap,lac)” Arch.Microbiol. 153 1-6
2.W.Metcalf, P.Steed & B.Wanner (1990) “Identification of Phosphate Starvation-Inducible Genes in Escherichia coli K-12 by DNA Sequence Analysis of psi::lacZ(Mu dl) Transcriptional Fusions” J.Bacteriol. 172 3191-3200
3.B.Castilho, P.Olfson & M.Casadaban (1984) “Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons” J.Bacteriol. 158 488-495

-- Eric Kofoid

cfu/pfu calculation worksheet

Monday, May 18th, 2009

While doing an experiment recently, I found that I was doing a lot of guesswork to figure out how much I should dilute resuspended colonies to get nicely spaced single colonies for viable counts. Since I was doing the simple calculations a lot, I decided to put together a worksheet to do it for me.

I also added a panel that does the calculation the other direction –from # of colonies on a plate back to the original # of cells in the original culture.

Trivial? Yes.

Useful? Hopefully.

Accurate? Probably.



-- Doug

First Solexa Data In!

Wednesday, April 1st, 2009

(For those of you who may have forgotten, Solexa sequencing is a rapid, highly automated method of generating millions of short sequences at random across a DNA sample — often, an entire genome).

I have just received the first set of Solexa data from our collaboration with Fritz Roth and his colleagues, Yong Lu & Joe Mellor. The image below shows “read depth” (the number of runs which cross a given point) in the neighborhood of lacZ for strains TT24815 and TT25790. We expect this measurement to increase in proportion to the degree of amplification. Coverage over non-amplified areas of the plasmid and chromosome exceeded 50-fold for both strains.

Small red arrows show my guess at the amplification endpoints. The TT24815 array stretches from approximately 138256 to 166250 (~28 KB), and TT25790,  from 131600 to 159300 (~28 KB), where the coordinates refer to our standard F’128 sequence counting clockwise from the first nucleotide of IS3A.

Strain TT25790 contains Elisabeth’s known inversion duplication array (EK568), for which we have sequenced a single join point (134075->134087 recombined into 132108<-132098). Small blue arrows show these two tracts. In our simple models of inverted duplication formation, join 1 forms from their recombination, either directly (“Flying Walendas”) or by assymetric deletions of a larger toxic structure (“Slytherin”). Furthermore, all Solexa data in the array should begin at the leftmost blue arrow, gratifyingly close to my guessed endpoint. Join point 2 will be defined by a sequence near the righthand red arrow and its inverted complement at a position yet to be found in the amplified region. I shall go hunting!

Strain TT24815 was chosen for its recalcitrant nature — we were never able to find any join points, but assumed for this reason that it was a likely candidate for an amplified inverted duplication, as crossover sites in these entities are truly difficult to locate and sequence. We were hoping to get two new bits of previously unknown information out of it. Once again, half of each of the join points should be defined by small sequence inversions in the neighborhood of the red arrows, assuming that this is a truly simple array of elements representing one kind of inverted duplication. More hunting!

If you use your browser’s zoom feature, you can inspect the image with better resolution. You can also download a detailed PDF file.


-- Eric Kofoid

Burnt by Prior Cassettes

Saturday, March 7th, 2009

For the second time, I have been burnt by the presence of a previously inserted cassette in a background I was using for linear transformation. In both cases, it was a pro::spec swap. I noticed an abnormally high number of camR transformants the next day. I printed the colonies to spectinomycin medium , and discovered that most were sensitive! What I had succeeded in swapping was the pre-existing cassette for the new one. Very few of the transformants were those which I had intended.

In parallel experiments, I found that the number of transformants increased over the course of a couple of days. Our standard UNI cassette (with the exception of tetAR) is a drug resistance gene embedded in an otherwise constant context, originally derived from the chloramphicol resistance locus of pACYC184. This means that sequence blocks about 200 bp at each end of the cassettes are always the same. Apparently, these regions are being used as recombination sites during the transformation.

I surmise that lambda red is not causing these insertions, but rather the host recombination system, because of the kinetics of their appearance. We incubate our transformations overnight at 42°C, specifically to eliminate pKD46 — this should confine all transformation to the first few minutes after electroporation. In addition, lambda red contains no mechanism for resecting the 3′ end of the transforming DNA back through the 40 bp intended homology block to make available the core of the cassette for strand invasion. This could only be done by the host after the red system had decayed or been diluted by growth,  freeing the cell from red repression of the host system.

If I had used tetAR, I would never have noticed this effect. The only homology blocks available are the UNI ends, too small to be used by the host recombination system, but still requiring host resection to be exposed, which could only happen after elimination of red.

-- Eric Kofoid

Effects of L-Arabinose on Growth

Friday, March 6th, 2009

I have traditionally used 20 mM L-Arabinose to induce lambda red on pKD46. I have also noticed that r-m+ strains grew exceptionally slow during linear transformation. I decided to trace the cause of this defect by comparing their growth along with that of wild type plus or minus ampicillin (used to hold the plasmid) and plus varying concentrations of L-Arabinose. Results: 20 mM L-Arabinose inhibits growth of the r-m+ strain and inhibits yield of both strains. Ampicillin exacerbates the growth inhibition of the r-m+ strain, but not of the wild type. Experiment and data.

-- Eric Kofoid

Host Restriction of UNI Cassettes

Friday, March 6th, 2009

Our UNI CamR, KanR & SpecR cassettes all contain sites for endogenous LT2 restriction endonucleases. I tested the efficiency of transformation of these cassettes into a wild-type background (TT22970) and an r-m+ background (TT22971). My results are consistent with the hypothesis that UNI CamR cassettes are degraded by wild type LT2 when introduced by electroporation. Test description and data.

-- Eric Kofoid