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).

Week 20: September 8- September 14

Post transformational staining

Gram and spore stains of Bacillus subtilis was desired to ensure that after transformation had occured the cells still gave a result that was expected. B. subtilis is a gram-positive bacteria, and when a gram-stain is performed on it a purple to dark blue colour is expected, where as Escherichia coli is gram-negative and would stain red to a pink colour. During a spore stain of B. subtilis the spores stain green and the cells stain pink. E. coli was used as a positive control to ensure that the stain worked, providing a colour that was expected, and that the bacteria was growing successfully in our lab. Two gram stains for each of E. coli, B. subtilis were performed, as well as two spore stains for B. subtilis were performed. Before the stains were performed new streak plates of the bacteria were madeto incubate at 37 ° C overnight, ensuring that the bacteria came from fresh colonies. The bacteriawere plated on regular LB agar plates for incubation. The spores for B. subtilis were not streaked, as Israt and Anna Fei were taking care of the spores by changing the water in which they were stored to keep themmaintained properly.

Plates:
1. E. coli GFP E0040 combined transform. July 21, 2014 LB-amp
2. E. coli GFP E0040 combined transform. July 21, 2014 LB-amp
3. B. subtilis comK strain. September 5, 2014 Erythromycin + Lincomycin
4. B. subtilis comK strain. September 5, 2014 Erythromycin + Lincomycin

Bacterial smear:

These newly streaked plates provided the colonies for the bacterial smear slides necessary for the staining procedure. Clean microscope slides were labeled according to the corresponding plates from which the colonies were taken, and a circle on the back of the slide was drawn to show the area in which the smear would be done. 2uL of distilled water was dropped in the circled area on the slides, this water was then used to spread the bacteria around in. The bacteria was spread using a sterilized, using a Bunsen burner, inoculation loop, before the bacteria was fixed by running the slide through the Bunsen burner flame a four times. Once the smear was dried the staining was able to be performed.

Gram stain:

The bacterial smear was flooded with Crystal Violet for one minute before it was rinsed with distilled water. It was then flooded with Lugol's Iodine for one minute before being rinsed again. The smear was then decolourized using alternating applications of 95% ethanol for ten second and then distilled water until the water running off of the slide runs clear. The smear was then counter-stained with Safrinin for one minute before one last rinse with distilled water, and being blotted with Kim-wipes to dry the slide.

These stains were performed for each plate, after which it was evident from the naked eye that the smears with E. coli were pink, and the smears with B. subtilis were dark purple. In order to analyze the smears further they were analyzed under a high-tech photo-microscope, for this cover slips were placed on the smears to protect both the bacterial smears and the microscope.

Spore stain:

Two bacterial smears were performed for the B. subtilis spores, before the stains were done. To collect the spores for the smear the suspended spores were centrifuged for two minutes at 14,000 rpm, so that the spores could be collected from the pellet. After the bacteria were fixed on to the slide and the smear was dried, the slides were placed onto a hot plate, at 150 ° C, for the Malachite Green staining. A piece of paper towel was placed over the smear so that it could be completely flooded and steamed with Malachite Green for five minutes. The smear was continuously damped with the stain for the entire five minutes. After the five minutes and the paper towel was removed the smear was rinsed with distilled water. The smear was then counter-stained with Safrinin for one minute before the final rinse and drying. The stain was visualized in the photo-microscope and a photo was taken using a mobile device as the microscope itself could not take coloured photos which were necessary to show the spores as green and the cells as not green.

Week 21: September 14 - September 20

Post sporulation and re-generation staining

Israt had plated regenerated B. subtilis on LB agar plates, which were incubated overnight at 37 ° C so that staining could be done on them to check to make sure the bacteria looked the same when analyzed as before they were sporulated. Six slides were made using Israt's plates, four for gram staining and two for spore staining.

Plates:
1. B. subtilis Spore. September 18, 2014
2. B. subtilis Spore+xylose. September 16, 2014

From these plates two slides for each was made for gram staining, and then two slides from Plate 1 for spore staining. The same gram and spore staining procedures, as above, was used for the slides, the only modification made to the staining procedure was that the counter-stain Safrinin was left on for one and half minutes instead of just one minute as it was noticed that the Safrinin was too light in colour in the previous staining slides.

From the naked eye the slides appeared to be successful, the gram stained slides were all purple and the spore stain slides were completely pink, but they will be analyzed at a later date under a microscope.

Week 22: September 21 - September 27

Post sporulation and re-generation imaging

Michael Wilton, University of Calgary Health Sciences Campus, allowed us to use the microscope in his lab to image the slide stains for the regenerated B. subtilis spores. All of the slides gave desired results:

Gram stain:

All slides gave a purple colour, showing that the bacteria still behaves as a gram=positive bacteria after sporulation and regeneration.

Spore stain:

All slides showed vastly more pink cells compared to green spores. This is good because it shows that the regeneration of spores has a high efficiency even when the spores are only grown on LB at 37 ° C overnight.

Desiccation of B. subtilis spores

We wanted to test if the spores could still be regenerated and generate viable bacteria after the spores were subject to harsh conditions. To do this, a collection of short term storage of spores (kept in distilled water at 4 ° C), and a collection of long term storage (kept in distilled water and frozen) were separated into five 0.5mL tubes, one for each condition. Each of these tubes was then centrifuged, for five minutes at 14 000 rpm, so that the distilled water that the spores were suspended in could be removed before the spores were subjected to each condition. The chosen conditions were designed to subject the spores to a wide range of conditions, some of which were chosen to simulate some of the conditions that the diagnostic tool might be subject to, such as excessive heat, while others were chosen to see what exactly the pores were able to survive through, such as chemical conditions. The five chosen conditions were; 1% bleach, 70% ethanol, complete drying, heating at 60 ° C, and heating at 90 ° C.

• 1% bleach: Centrifuged spores re-suspended in 50uL 1% bleach.
• 70% ethanol: Centrifuged spores re-suspended in 50uL 70% ethanol.
• Drying: Centrifuged spores were completely dried in a Vacufuge at 30 ° C for 10 minutes.
• Heating at 60 ° C: Centrifuged spores were placed in a hot water bath at 60 ° C.
• Heating at 90 ° C: Centrifuged spores were placed on a hot plate at 90 ° C.

The spores were subjected to each of these conditions, at 5:35 pm on September 25, 2014, and left for 24 hours before they were re-suspended in 50uL of distilled water. From this solution the spores were centrifuged to separate spores from the supernatant so that the spores could be spread on LB agar overnight to see if the spores were able to regenerate and cultures were able to grow. This allowed for quantitative data to be taken of the cultures to see which conditions proved to be the hardest on the spores.

Week 22: September 22 - September 28

Sporulation with xylose

It was decided that sporulation of B. subtilis should be done but with using xylose during different times during the sporulation process rather than just in the media for regeneration to test if this made the spores more viable after regeneration had occured. The initial culture and addition of xylose (to end up with 1% xylose in culture) to B. subtilis was done by Israt and I added xylose when the culture was added to the SGx2 media before being shaken for 2-3 days. Unfortunately, I miss interpreted Israt's instructions and put xylose in both cultures, and not just in the one that did not already have xylose in it. Because of this, is was determined that we would re-do the experiment after making a new batch of xylose (Israt) and SGx2 media (Shelby).

Week 23: September 29 - October 5

Xylose sporulation and SGx2 media

SGx2 media was made for the sporulation process, the recipe was originally obtained from Anna Fei, and was modified for a smaller volume, as the media tends to get cloudy quickly and then can no longer be used. The original recipe was for 500 mL total volume and was reduced to 250 mL for our purposes.

Original recipe:

1. 500 mL of distilled water
2. 8 g Difo nutrient broth
3. 1 g KCl
4. 0.5 g MgSO4 • 7H2O
5. Autoclave above mixture before adding the remaining ingredients
6. 0.5 mL 1 M Ca(NO3)2
7. 0.5 mL 0.1 M MnCl2 • 4H2O
8. 0.5 mL 1 mM FeSO4
9. 1 mL 50% Glucose (filtered)

This recipe was cut in half and was finished being made on September 30, 2014, the same day that culture and xylose was added for the 2-3 day shaking process.

A new batch of B. subtilis was cultured and a new batch of 10% xylose were made by Israt in preparation for the xylose sporulation of B. subtilis. She also added xylose to one of the cultures before shaking for 6-8 hours, resulting in a 1% xylose concentration in the culture, and left the other with out xylose so that it could be added when SGx2 media was added. After being shaken, 0.125mL of culture was added to a volume of 25 mL of SGx2 media, and 2.5 mL of 10% xylose was added to the tube not containing xylose already. This meant that both tubes had a concentration of 1% xylose in the end. These were then shaken for 3 days before being placed in the fridge in preparation for spore removal.

B. subtilis spore treatment results:

There was growth seen for each condition, with exception to the spores being lit on fire in the Bunsen burner which was expected. Overall, the long term storage spores, frozen, seemed to better survive the chemical conditions, and the short term, fridge, spores seemed to better survive the extreme temperature conditions. For long term storage, there was 3 large colonies seen on 1% Bleach plate, 5 large colonies on 70% ethanol, and 11 large colonies on drying. Whereas, for short term storage, there was 2 large and 1 small colonies on 70% ethanol, 1 medium colony on 60 ° C, and 1 medium colony on 90 ° C plate. It was later decided that the long term storage spores should be plated by themselves, without them being subject to any other conditions. This resulted in a full lawn of large colonies after being incubated at 37 ° C for 72 hours. From this it appears that long term storage is a better method for preserving the B. subtilis spores than short term storage for the purposes of our diagnostic device.

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
• Huddleston, WR and Cusack, F. (2014). Cellular, Molecular and Microbial Biology 343. Calgary: University of Calgary (pp. Ap4-1 - Ap4-2). Calgary, Canada: University of Calgary.
• 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