Defining the link between enterotoxin production and sporulation in C. perfringens

The second most common cause of bacterial foodborne illness is Clostridium perfringens type A. These isolates produce an enterotoxin (CPE), and an estimated 250,000 cases of resultant food poisoning occur annually in the U.S. Forty years ago, it was postulated that sporulation and enterotoxin production were linked and, in fact, C. perfringens type A isolates only produce CPE during sporulation.

Four sigma factors mediate sporulation in C. perfringens; however, the exact roles of two of them (SigF and SigG) are unknown. After confirming that sporulating wild-type SM101 cultures produce SigF and SigG, Li and McClane prepared isogenic sigF or sigG null mutants. They used the MasterPure™ Gram-Positive DNA Purification Kit to isolate DNA for Southern blotting, to confirm the presence of a single intron insertion in the SM101::sigF and SM101::sigG mutants. The detection of alternative sigma factor production by Western blot analysis was aided by Ready-Lyse™ Lysozyme. The authors concluded that all four sigma factors are needed for sporulation, but only SigE, SigF, and SigK are needed for synthesis of CPE. The results of this study indicate a previously unappreciated level of complexity for the regulation of cpe transcription.

Li, J. and McClane, B. (2010). Evaluating the Involvement of Alternative Sigma Factors SigF and SigG in Clostridium perfringens Sporulation and Enterotoxin Synthesis Infect. Immun. : 10.1128/IAI.00528-10

Using Nextera technology for RNA-Seq expression analysis

We’ve had several inquiries about using Nextera™ technology with cDNA, in order to generate libraries for RNA-Seq and subsequent gene expression analysis. We are grateful to the Richard M. Myers Laboratory at the Hudson Alpha Institute for Biotechnology (Huntsville, AL), for testing this application of Nextera technology. They used the following strategy (click on all figures to enlarge them):

Strategy for library preparation

With this strategy, decreased coverage at the ends of cDNAs is expected because two transposome insertions are required to produce a functional sequencing template.

Libraries using human RNA (ECC-1) were prepared with both the Nextera workflow and a standard RNA-Seq workflow, and sequenced on the Illumina GAII platform. Two Nextera libraries were prepared: one from 50 ng of cDNA, and the other from only 10 ng of cDNA. The results from both the conventional RNA-Seq and Nextera procedures compare very well:

Relative gene expression analysis

The data show that Nextera technology yields representative libraries, even with very low amounts of cDNA:

Correlation based on sample input amount

As seen from the coverage of the cDNA termini and library complexity, both libraries were highly complex and representative of the transcripts. The red arrows show the expected drop in coverage of 5' and 3’ ends with the Nextera library:

Library coverage and complexity

Purification of DNA and RNA from the same sample using the MasterPure Complete Kit

Many customers have asked how they can process both DNA and RNA from the same sample, using the MasterPure™ Complete DNA and RNA Purification Kit. The following protocol has been validated, and successfully used by customers:

1. Follow the protocol for total nucleic acids (TNA) in the product literature, including Lysis (Part A) and the first two steps for Precipitation (Part B, steps 1 and 2).

At this point, split the supernatant into two equal portions. Label one tube “DNA” and the other “RNA”.

1. Add 250 µl of isopropanol to both tubes, invert 30-40 times to mix.
2. Centrifuge the tubes for 10 minutes at maximum speed in a microcentrifuge at 4°C.
3. Carefully remove the isopropanol with a pipet, taking care not to dislodge the pellet.
4. Rinse with 70% ethanol. Centrifuge briefly if the pellet is dislodged. Remove residual ethanol with a pipet.

DNA Protocol (for the tube marked “DNA”)

1. Resuspend the pellet in 100 µl TE Buffer.
2. Add 1 µl of RNase A to sample and mix well.
3. Incubate @ 37°C for 10 minutes. Note: Additional incubation (up to 30 minutes) may be needed.
4. Add 100 µl of 2X T&C Lysis Solution, and mix by vortexing for 5 seconds.
5. Add 100 µl of MPC protein precipitation reagent, mix by vortexing for 10 seconds. Place on ice for 3-5 minutes.
6. Centrifuge for 10 minutes at ≥10,000 x g.
7. Transfer the supernatant to a new microcentrifuge tube and discard the pellet.
8. Add 250 µl of isopropanol. Invert 30-40 times to mix.
9. Centrifuge the tubes for 10 minutes at maximum speed in a microcentrifuge at 4°C.
10. Carefully remove the isopropanol with a pipet, taking care not to dislodge the pellet.
11. Rinse twice with 70% ethanol. Centrifuge briefly if the pellet is dislodged. Remove all residual ethanol with a pipet.
12. Resuspend the DNA in 10-35 µl of TE Buffer.

RNA Protocol (for the tube marked “RNA”)

1. Prepare 100 µl of DNase I solution by diluting 2.5 µl of RNase-Free DNase I up to 100 µl with 1X DNase buffer.
2. Resuspend the pellet in 100 µl of DNase I solution.
3. Continue with steps 3 through 12 from the DNA Protocol (above).

Ribo-Zero rRNA removal and gene expression analysis of fragmented RNA

A significant advantage of the Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) is that it can remove virtually all the rRNA, even from degraded total RNA samples. We examined correlation of gene expression between RNA-Seq libraries prepared from intact and fragmented RNA samples treated with the Ribo-Zero Kit, as well as a competitive kit.

Intact and partially fragmented Universal Human Reference RNA (UHRR) samples (2 x 2.5 µg each) were treated with either the Ribo-Zero Kit or a competitive rRNA removal kit. The respective rRNA-depleted samples were pooled and, for each, RNA-Seq libraries were prepared in triplicate from the equivalent of 1 µg total RNA, using a random-primed cDNA synthesis method. Replicates of the respective RNA-Seq libraries were pooled and sequencing was performed using an Illumina GAIIx sequencer with 36-nt reads. The data were analyzed using Illumina’s Pipeline Eland_rna Module and CASAVA Software.

We observed a higher correlation (R² = 0.9396) in genes detected between intact and fragmented Ribo-Zero rRNA-depleted RNA-Seq libraries (A) compared to the corresponding RNA-Seq libraries prepared using the competitive kit (R² = 0.8940) (B). Also, an additional 1,016 genes were mapped (with ≥10 reads) for the Ribo-Zero RNA-Seq libraries, indicating better coverage of transcripts with reduced rRNA background.

A. Ribo-Zero rRNA-depleted RNA-Seq libraries

B. Competitor rRNA-depleted RNA-Seq libraries

Fosmid cloning enables new techniques in synthetic biology

In a recent functional genomics study, Sommer et al. cite the use of the CopyControl™ Fosmid Library Production Kit to create a library from plant biomass DNA. Plant biomass is being explored for use in new biofuel development, in an effort to discover genetic functionalities that will allow growth improvement in key microbes by overcoming toxic/inhibitory compounds that are byproducts of biofuel conversions. Clones harboring these fosmids were tested against seven known growth inhibitors from three chemical groups (alcohols, aldehydes, and organic acids). The authors located genetic functionalities on two fosmids that improved the growth of the E. coli host cell by 5.7- and 6.9-fold in the presence of these inhibitory compounds. They then produced chimeric clones that contained all of the desired chemical functionalities into a three-gene construct that confers improved tolerance for these inhibitor compounds. The information gleaned from this study will be useful for scientists who wish to develop strains of bacteria that can generate new biofuels more efficiently and for longer periods of time.

Other Epicentre products cited include the End-It™ DNA End-Repair Kit for end-polishing of size-selected, gel-purified DNA; and the FosmidMAX™ DNA Purification Kit to purify DNA from the metagenomic fosmid clones.

Sommer, M. et al. (2010). A functional metagenomic approach for expanding the synthetic biology toolbox for biomass conversion Molecular Systems Biology, 6 DOI: 10.1038/msb.2010.16