Transformers' Journal

Week 1: May 1-2

Our first week consisted mainly of formal introductions and various brainstorming sessions regarding the direction of the 2014 project. The options were narrowed down to a biofilm based water filtration system and a rapid, multi-diagnostic test. Experts in the field of bioremediation, molecular biology, and medicine were consulted in order to gain more insight on the prospects of both project ideas. Ultimately, the rapid multi-diagnostic was voted over the water filtration system and we began preliminary planning.

Week 2: May 5-9

We attended a series of workshops tailored towards those who are researching various aspects of molecular biology. We learned the basics of genetic engineering and lab protocol in preparation for future research. These workshops were especially helpful for our team members who had no prior experience working in a lab.

Week 3: May 12-16

The techniques we learned during the workshops were applied within our own lab; we made stocks of competent E. coli., poured anti-bacterial agar, performed plasmid mini-prep, ran gels, etc.. The team decided upon an in-vivo diagnostic system that could detect pathogens through homologous recombination. Literature research was conducted on the subject of how to enhance the natural transformation of E. coli.

In regards to E. coli., it was discovered that E. coli. was closely related to H. influenzae, a species of bacteria well known for it's high frequencies of natural transformation (competency). H. influenzae uses a Type-IV pilus structure to uptake DNA, so the team researched whether or not such a system could be implemented into E. coli. to yield similar transformation rates. Studies conducted by Dr. Rosemary Redfield - an E. coli. expert - showed that all gram-negative bacteria share two common regulator genes for competency; sxy and CRP (Sinha & Redfield, 2012). A deeper analysis of Dr. Redfield's papers also revealed that E. coli. lacks a pilF2 homologue. Curious, we contacted Dr. Redfield to gain insight into the subject. Dr. Redfield stated that during her experiments, all the proteins involved in DNA uptake were able to be expressed, but the rates of competency in E. coli. were quite low to begin with. It was decided that E. coli. would not be used for our project as there were many unknown variables which dictated the expression of competence gene homologues in E. coli.

Additional studies on E. coli. conducted by Sun et. al. were looked at. The findings of these studies stated that OmpA (an outer membrane protein) plays a role in uptaking DNA from conjugative plasmids and bacteriophages (Sun, Wang, Chen, & Zhan, 2013). The researchers also hypothesized that OmpA plays a stimulatory role in DNA uptake in liquid Ca2+ cultures, along with an inhibitory role on agar plates. (Sun et al., 2013) OmpA negative strains (JW0940) were found to have a higher transformation frequency than wild-type strains (BW25113) when grown on agar (Sun et al., 2013). Surprisingly, transformation frequencies did not change with varying agar thickness, so it remains uncertain how agar plays a role in E. coli.'s natural transformation (Sun et al., 2013). Conversely, the OmpA negative strains showed a lower frequency of transformation than wild-type strains when grown in liquid Ca2+ cultures (Sun et al., 2013). We concluded that it may be possible to make E. coli. naturally competent on agar by replacing the OmpA gene with a gene that codes for kanamycin resistance.

In additional to E. coli., B. subtilis. was researched as a potential platform for DNA uptake. Current trends and methods in B. subtilis transformation were analyzed, with competency rates ranging from 104 to 107 transformants per microgram (Zhang & Zhang, 2013). One method utilized xylose in conjunction with the pAX01-comK plasmid to overexpress the competency regulator gene (ComK) in B. subtilis and create "supercompetent cells" (Zhang & Zhang, 2013). This yielded the highest transformation rate among the methods studied (Zhang & Zhang, 2013).

Other members of our team developed the basis for a "genetic circuit" which could detect the DNA of pathogens upon uptake by a competent bacterial cell.

Works Cited:

Eichenberger, P. (2012). Genomics and cellular biology of endospore formation. In P. Graumann (Ed.), Bacillus : cellular and molecular biology (pp. 319 - 350). Norfolk, UK: Caister Academic Press

Graumann, P. (2012). Preface. In P. Graumann (Ed.), Bacillus : cellular and molecular biology (pp. 319 - 350). Norfolk, UK: Caister Academic Press

Maier, B. (2012). Competence and transformation. In P. Graumann (Ed.), Bacillus : cellular and molecular biology (pp. 319 - 350). Norfolk, UK: Caister Academic Press

Sinha, S., & Redfield, R.J. (2012). Natural DNA uptake by Escherichia coli. PLoS One, 7(4), e35620.

Sun, D., Wang, B., Chen, M., & Zhan, L. (2013). Block and boost DNA transfer: opposite roles of OmpA in natural and artificial transformation of Escherichia coli. PLoS One, 8(3):e59019.

Zhang, X-Z., & Zhang Y-H. (2011). Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis. Microbial Biotechnology, 4(1):98-105