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.

Week 4: May 20-23

Literature searches were conducted on the regulation of competence in B. subtilis. We contacted Dr. Zhang and requested the pAX01-comK plasmid, also making sure to ask if only certain strains of B. subtilis can be made supercompetent. Dr. Zhang stated that any 168 derivative strain could be made supercompetent and told us to request the necessary plasmid from the Bacillus Genetic Stock Centre. Further literature searches were conducted to find the best plasmids to induce competency in B. subtilis. A highly experienced B. subtilis researcher at the University of Calgary, Dr. Wong, was contacted in order to gain more information on B. subtilis and hopefully acquire a supercompetent B. subtilis strain.

Following the decision to use B. subtilis as the platform for our in-vivo diagnostic tool, we officially divided our team into two groups: the Transformers and the Detectives. The goal of the former is to facilitate high transformation rates in B. subtilis, while the goal of the latter is to develop and tweak the pathogen detection system.

Week 5: May 25-30

We attended the Alberta Innovates Technology Futures workshops hosted by geekStarter in order to learn more about policy & practices, presentation skills, current trends in synthetic biology, and 3D digital modelling. Speakers included Kelly Drinkwater of iGEM, Cesar Rodriguez of Autodesk, and David Lloyd of FredSense. Following the presentations, the speakers provided useful feedback on our project from both a scientific and human practices perspective. We also met the Lethbridge iGEM team and was introduced to their project.

Later in the week, we prepared overnight cultures and plasmid mini-preps for the parts required to assemble the genetic circuit. We also created antibiotic solutions for spectinomycin, erythromycin, and lincomycin in preparation for the supercompetent B. subtilis colonies we were going to receive, along with future use. The B. subtilis strains were generously donated to us by Dr. Wong and Dr. Wu of the University of Calgary. Given the importance of these strains, we immediately prepared backup stocks of the received colonies and incubated them. Meanwhile, colony PCR was performed on E. coli. colonies containing tetR+RBS and the 5`integration, which we prepared beforehand. Dr. Wong recommended we do additional background research on the life cycle and biology of B. subtilis.

We learned that B. subtilis, like most bacteria, are dependent on quorum sensing when trying to induce competence. When B. subtilis cells cluster together and their cell density increases, a collection of small peptides (called competence hormones) are excreted from each cell and recognized by adjacent cells. Research shows that the cells will only become competent if the concentration of these peptides is quite high (Graumann, 2012). Research was also done on sporulation, a process by which bacteria enter a dormant state in order to survive adverse conditions such as starvation, irradiation, and heat (Eichenberger, 2012). As B. subtilis reach the stationary phase of their life cycle, some cells acquire competence while others sporulate (Maier, 2012). We determined that B. subtilis' tendency to sporulate under extreme conditions may be beneficial for our purposes, as the durability of a B. subtilis spore would facilitate an easy transportation of our diagnostic tool around the world. In theory, the spores would not require refrigeration en-route to their destination and could be stored for significantly long periods of time.

In regards to sporulation, the mother cell and the foreshore are genetically identical, but certain proteins must be made specifically in the developing spore. Thus, certain set of genes transcribed from mother cell DNA must differ from the set transcribed from foreshore DNA. The sporulation sigma factors replace the principal negative cell sigma factor A in RNA polymerase holoenzyme - possibly by out competing sigmaA for RNA polymerase (Eichenberger, 2012).

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