Thursday, April 29, 2010

Identification of suitable hosts for small-molecule functional metagenomics

The majority of soil-dwelling bacteria cannot be cultured with standard microbial culture methods, and represent an untapped reservoir of novel small molecules that are key components of biosynthetic pathways. Functional metagenomics is one approach to solving this problem; however, screening methods are limited by their dependence on a host organism to facilitate the expression of genes from environmental DNA (eDNA).  To overcome the limitations of relying on a single host organism, Craig et al.* examined six unique Proteobacteria hosts for functional metagenomic screening. To construct  soil eDNA libraries, they blunt-ended the isolated DNA with the End-It™ DNA End-Repair Kit, ligated it into a cosmid vector, packaged the DNA using MaxPlax™ Lambda Packaging Extracts, and transformed TransforMax™ EC100 Electrocompetent E. coli. The eDNA libraries were then used to transform each of the six hosts being studied, and the resulting libraries were screened for antibacterial activity, or altered pigment or morphology. Cosmid DNA from selected colonies was electroporated into TransforMax™ EPI300 Electrocompetent E. coli, and each cosmid clone was analyzed by 454 sequencing.

From this initial broad-host-range functional metagenomic study, Craig et al. demonstrate that some surrogate expression hosts will be better suited to using foreign genetic material than others. They conclude that continued exploration of phylogenetically diverse bacteria as hosts for functional metagenomic screening is likely to identify additional strains that will be suitable for eDNA functional metagenomics.


ResearchBlogging.org*Craig, J. et al. (2010). Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria Applied and Environmental Microbiology, 76 (5), 1633-1641 DOI: 10.1128/AEM.02169-09

Tuesday, April 27, 2010

Labeling RNA for gene expression analysis

Here are answers to questions that we commonly receive regarding the TargetAmp™ Antisense RNA (aRNA) Amplification Kits.

Can I use the TargetAmp Kits for producing fluorescent-labeled aRNA?
Yes. The TargetAmp 1-Round and the TargetAmp 2-Round Aminoallyl-aRNA Amplification Kits produce aminoallyl-aRNA (AA-aRNA), which can be readily labeled with a fluorescent dye conjugated to N-hydroxysuccinimide (NHS). 

What are the advantages of aminoallyl-based (indirect) labeling over direct incorporation of a labeled NTP?
Aminoallyl-UTP is more efficiently incorporated into the aRNA during the in vitro transcription reaction, compared to labeled nucleotides. Additionally, the conjugation of an amine-reactive NHS ester of biotin or a fluorescent dye to AA-aRNA can be a less expensive method to label the target compared to direct incorporation of labeled nucleotides. 

What are the advantages of direct labeling of aRNA?
Labeling by direct incorporation is faster compared to the aminoallyl-based procedure. Conjugation of AA-aRNA to a dye- or biotin-NHS ester after in vitro transcription requires a 1-hour incubation, and an additional clean-up step, compared to direct incorporation. Further, labeling aRNA by direct incorporation does not require the use of toxic reagents (such as DMSO), which are used in the indirect labeling protocol.

To find the TargetAmp Kit that’s right for your application, please see our selection guide.

Friday, April 23, 2010

EZ-Tn5™ Transposomes help reveal virulence factors in Acinetobacter baumanii

Acinetobacter baumanii is a pathogenic bacterium that has been demonstrated to cause pneumonia, skin infections, and secondary meningitis, predominantly in a health-care facility setting. Its ability to form biofilms on inert surfaces is instrumental in creating reservoirs for opportunistic infection. Unfortunately, very little is known about the genetics of this organism, which hinders the study of pathogenic factors in A. baumanii infection. In a recent study, Jacobs et al.* describe the use of the EZ-Tn5™ Transposome in inactivation of the Phospholipase D gene in various strains of A. baumanii, and report that the creation of a polar mutation in this locus reduces the pathogenicity of the bacterium. Inactivation of Phospholipase D showed reduction of overall A. baumanii bioburden in the blood, heart, and liver in a murine model, but did not reduce the bioburden in the lungs. The utility of the EZ-Tn5 Transposome system to develop genetic systems for novel or poorly studied microbes is once again demonstrated.

ResearchBlogging.org*Jacobs, A. et al. (2010). Inactivation of Phospholipase D Diminishes Acinetobacter baumannii Pathogenesis Infection and Immunity, 78 (5), 1952-1962 DOI: 10.1128/IAI.00889-09

Tuesday, April 20, 2010

Complete Genomics sequencing platform built on Epicentre’s enzymes

Last November, Complete Genomics published details of its third-generation sequencing platform that promises extremely high throughput at an estimated per-library cost of $4,400 for consumables.* The researchers sequenced three human genomes, generating an average of 45- to 87-fold coverage per genome and identifying 3.2 to 4.5 million sequence variants per genome.

The combinatorial probe-anchor ligation (cPAL) chemistry relies on the formation of DNA concatamers (termed “nanoballs”) that are clonally amplified from circular templates (see Complete Genomics workflow summary). The templates are prepared using Epicentre’s CircLigase ssDNA Ligase. In addition, the library preparation procedure used:
ResearchBlogging.org
*Drmanac, R. et al. (2009). Human Genome Sequencing Using Unchained Base Reads on Self-Assembling DNA Nanoarrays Science, 327 (5961), 78-81 DOI: 10.1126/science.1181498

Thursday, April 15, 2010

FailSafe™ PCR System earns praise

It's always nice when customers appreciate our products. It's even better when they make a video telling the world about why the product performed so well.

Alan M at Benchfly describes his experience with FailSafe™ Buffer D in the clip below. No, we didn't pay him a dime :) We appreciate his comments and, of course, we agree with his assessment of why the FailSafe System is the best product for PCR of challenging and routine templates. Don't just take our word for it, though--the FailSafe PCR System is supported by over 100 publications.

Note: We recommend that customers always optimize their PCR conditions for each new template, using the FailSafe PCR PreMix Selection Kit.

Tuesday, April 13, 2010

CHI XGen Congress session on Nextera™ technology

Epicentre presented a breakfast session on Nextera™ technology and its applications at the CHI XGen Congress (March 15-19, 2010). We are pleased to make a video recording of the session available through SciVee. The presentation is approximately 27 minutes long.

Friday, April 9, 2010

Save big on library prep for Roche GS FLX

As many users of Roche 454 sequencers have moved to the newer GS FLX Titanium chemistry, we are now offering big discounts on Nextera™ DNA Sample Prep Kits for the original GS FLX chemistry (Cat. # FL09115 and FL091120).

Let us know if you’re using the original GS FLX reagents by leaving a comment here. If you’d like to try the Nextera library prep method—which offers a streamlined, rapid workflow and low input DNA requirements—this is your chance to save money as well as time. Please contact us by e-mail to obtain a discounted price: Nextera {at} epibio {dot} com

Tuesday, April 6, 2010

Transposomics made EZ

EPICENTRE’s EZ-Tn5™ Transposomes (the synaptic complex between  transposon DNA and the hyperactive EZ-Tn5 Transposase) have been used for generating bacterial mutation libraries, strain development, and creating knockout/knock-ins since 1999. The easy-to use Transposomes have been used in the development of transposition libraries in over 65 microbial species (both Gram-positive and Gram-negative), and even in yeast and trypanosomes. Our citations page lists publications grouped by the organism studied.

The ready-to-use Transposomes contain kanamycin- or trimethoprim-resistance cassettes. Additional Transposomes can be created easily, using simple molecular techniques  with the EZ-Tn5 pMOD series of transposon construction vectors and hyperactive EZ-Tn5 Transposase. We also provide a comprehensive introduction to transposon methods and product selection guide, and additional documentation (114K PDF) for the preparation and use of custom Transposomes in many bacteria.