Team:OLS Canmore/Results

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RESULTS

Throughout the year we performed various different assays and working out of our small lab experienced a few technical difficulties especially with minipreps. However, by the end of the year we were able to submit two new biobrick parts! We also have some promising proof of concept results to show that these biobricks work as expected. Below the results of all our experiments are outlined.

Because we did not have the biobrick version of our parts completed for a portion of the year, we chose to conduct some experiments with the promoter and protein coding sequences inside a pUC57 backbone. We obtained theses plasmids in a pUC57 vector through gene synthesis. Or so we thought.

On October 13th, after many inconsistent results in the lab, we realized by looking at the original tube of synthesized DNA that it was mistakenly placed in a pet vector(Kanamycin resistance) instead of pUC57 (Ampicillin resistance). This was only labelled on the tube and not on any paperwork or any communication (that was all labelled pUC57). We had ordered them in pUC57 and they had confirmed this. The mistake is a random, yet unfortunate, accident. Despite this, we were able to push some lab work into the last week before the wiki freeze and are able to share some promising results and submit two biobrick parts.

The error in antibiotic use explains why we were able to get results immediately after transforming the non-biobrick plasmids, but were not able to maintain the cell lines.

Summary:

• Two biobrick parts have been submitted to the iGEM registry. These are the two composite keratinase parts. Because of the synthesis mistake, characterization is ongoing and we hope to have some preliminary results to share in our presentation in Boston.
• Keratinases seem to be possibly toxic to E.coli. KerA seems to be more toxic than KerUS. In the future, expression cassettes of the keratinases should be created using a promoter that stimulates lower rates of expression and that is less “leaky”.
• The non-biobrick KerUS plasmid seems promising and in several experiments shows evidence of some enzyme activity. Seen either as clearing on skim milk plates, or possible leaching of protein-based dye from the cap of the falcon tubes.
• Our modified miniprep seems to skirt many of the problems experienced in our lab.
• The biobrick KerUS plasmid seems to have been unsuccessfully created because they results obtained do not show evidence of enzyme activity. However, gel electrophoresis using miniprepped biobrick KerUS plasmid are also unsuccessful. This points to a problem with the creation of the part, and not with the expression of keratinase.

Creation of the biobrick parts:
After starting from scratch, knowing that the synthesized parts were mistakenly placed in a backbone with Kanamycin resistance, we had no issue growing up and maintaining cell cultures and plates. However, because of the time crunch between the 13th of October and the part submission deadline on the 21st, we were helped by our mentor Lisa Oberding. She works for Fredsense and has full-time access to a lab, therefore she was able to assist in creating the parts. She also set up some assay with the biobrick parts in her lab, while we ran them concurrently at our school. We are very grateful to have her as our mentor, otherwise we would never have been able to submit parts.

Starting From Scratch:
The description of the results is quite vague because they were only obtained a few hours before the wiki freeze. We would love to discuss these during the poster session, or through email and facebook (contact info at the bottom of the page)! Also, we’ll be sure to mention these during our presentation.

Starting from the beginning, the synthesized plasmids were transformed and plated onto Kanamycin plates. Unlike when we were transforming onto Ampicillin plates, the cultures grew quicker and better.

Then a restriction digest and gel electrophoresis were run in order to be able to construct the biobrick. The gel below shows the, in the top row KerA digests, and in the bottom row, KerUS digests. For the KerA wells, 3 of the digests were positive showing a band at around 5,300bp for the vector (pET28b+), and approximately 1,400 bp KerUS and 1,100 bp for KerA. A ligation using these digested plasmids was done to assemble the composite keratinase parts into the pSB1C3 backbone. The ligation was plated with a red-white screen. On the pictures of the plate below you can see many white colonies, and only a few red.

A second miniprep, digest, and gel was conducted using the transformant cells. The two rows. The entire first row shows potential KerA colonies, and the second row shows entire KerUS colonies.

Characterization: We have set up our own independent assays, and Lisa (our mentor) has been replicating these in her lab. She had time to begin these 15 hours before us so the results shared below are hers. Our tests are still running and results will be incorporated into our presentation and shared during the poster sessions. The test conducted was the ‘Semi-quantitative Hair Degradation Assay’. The changes made were to take out the weighing step to make it a fully qualitative assay, and to incubate at two different temperatures. The feathers used were purple so clouder and more purple cultures would mean keratin degradation. The pictures of the initial cultures are shown below

1. KerA cultures grown overnight and induced with 0.1 or 0.5 mM final IPTG.
2. KerUS cultures grown overnight and induced with 0.1 or 0.5 mM final IPTG.
3. DH5alpha with C003 (which is a different plasmid with no keratinase genes), and with no IPTG.
4. Close-up of what the cultures looked like at t=0

After growing for 18 hours this is what the cultures looked like (shown above).

1. (Left to right) KerA 0.1mM IPTG, KerA 0.5mM IPTG, and control at 37 degrees Celsius.
2. (Left to right) KerUS 0.1mM IPTG, KerUS 0.5mM IPTG, and control at 37 degrees Celsius.
3. KerA 0.1 mM IPTG, KerA 0.5mM IPTG, and control at 30 degrees Celsius.
4. KerUS 0.1 mM IPTG, KerUS 0.5mM IPTG, and control at 30 degrees Celsius.

At 37 degrees Celsius there is not a very large difference between the control and the cells. This is either from the dye coming (dye bleed) off the feathers at this temperature, or this means the cells can actually all break down the feathers as at this temperature(E.coli may have natural proteases active). This is unsure as no obvious feather breakdown can be seen in the cultures yet.

At 30 degrees Celsius, both cultures containing the Keratinase genes induced with 0.1mM IPTG are more purple than the 0.5mM and the control. It appears that at 30 degrees there is not the same problem with the dye of the feathers bleeding as there is more of a difference between the control and keratinase cultures.

While there is no obvious feather degradation the results are still promising because they were only taken after 18 hours and contained a lot of feathers. We will continue to monitor the cultures over the coming days.


Toxicity:
There seems to be a toxicity issue with expressing keratinase in E.coli. This is inferred because of slow growth rate, low OD600 readings, and trouble culturing cells. This issue is more pronounced with the KerA gene and this after transforming the plasmids containing the promoter (that is a bit “leaky”) and KerA coding sequence, we were unable to plate any colonies. Therefore, all experiments were conducted using KerUS as it appears to be less toxic.

In the future however, we would like to create a plasmid with the Keratinase coding sequences downstream from a weaker and less “leaky” promoter. Cell free protein synthesis is also a technique that could be more effective to yield high amounts of keratinase while keeping the cells alive.

These results were also collected before discovering the mistake of antibiotic resistance. The slow growth seems more likely to be caused by growing in the wrong antibiotic. But, because of keratinases strong proteolytic activities, it is still likely that high expression could be toxic to E.coli.


Cell Lysis:
The protocol can be found on our ‘Experiments’ page.

The supernatant of the cell lysis was plated on skim milk plates, but due to our inability to use many of the reagents and chemicals commonly used to lyse cells the protocol had low success. The supernatant on skim milk plates (shown in the section below) show some cultures still growing indicating the the lysis was not wholly successful. The supernatant was also fairly viscous and this made it difficult to load into a gel for SDS-page.

Considerations for future experiments: Alternative methods of lysis could be used, such as sonication. However we do not have access to the necessary facilities for sonication, but in the future we would seek to partner with a university in the area that does. A second alternative and more feasible alternative is more repeated freeze-thaw steps.


Skim Milk Plates: The protocol can be found on our ‘Experiments’ page.

Non-Biobrick: The plates containing the supernatant of the lysis showed some zones of clearing. However this appeared to be due to colonies that grew on the plates as the lysis was not wholly successful. The plates with the plated cell culture showed zones of clearing around colonies with the KerUS plasmid. No zones of clearing were seen around colonies of DH5α without the keratinase-producing plasmids. However, for the non-lysed cells, the plates were kept on a slightly uneven angle so some spreading of the cultures can be seen. This seems to imply the production and secretion of keratinaseUS.

Old Biobrick (before synthesis realization): No zones of clearing were seen around any of the supernatants or any of the cultures. This could be due to the fact that the earlier non-biobrick cell lysis was conducted in a different lab with using liquid nitrogen, but in our school lab we could only use a longer incubation on ice. Or because the restriction digest and ligation of the part into psb1C3 was unsuccessful.

Considerations for future experiments: We were only able to conduct a limited amount of replicates and in the future would certainly like to repeat this assay to obtain more conclusive results.


Semi-quantitative Hair Degradation Assay:
The protocol can be found on our ‘Experiments’ page.

Non-Biobrick: No significant change in the mass of the hair was observed. However some discoloration of the falcon tubes used can be seen. The discoloration appears to be leaching of the dye in the cap. We have not been able to find more information of the dye used in the plastic cap but if any of the materials are protein based it could be a result of enzyme activity. It is important to note that the discoloration is only observed in tubes with KerUS cultures, and not in the control DH5α cultures.
The lack of visible hair degradation could be due to the low concentration of KerUS actually present in the culture compared to the amount of hair in the tube. It could also be a result of non-optimal temperature and pH for KerUS activity.

’Old’ Biobrick: For this assay we decided not to weigh the hair and feathers. Instead we used more pictures instead to document changes. No hair or feather degradation could be observed in any of the tubes. There was also no discoloration. This, much like the skim milk plate assays, tends to suggest an issue with the creation of the biobrick parts.

Considerations for future experiments: We would like to conduct more replicates of these experiments. Future considerations also include replicating the experiments with a higher concentration of KerUS, and optimal pH and temperature for KerUS activity.
As a supplementary control, the experiment should also be replicated with no hair to determine whether the discoloration is truly due to the plastic cap and not the hair.


Miniprep:
The protocol can be found on our ‘Experiments’ page.
During a lab weekend hosted by Geekstarter and the University of Lethbridge we were able to miniprep and run a gel to confirm the transformation of the non-biobrick KerUS plasmids. The gel shows a plasmid band at the the same size for all KerUS miniprepped samples, and suggests a successful transformation.

Modified: When it came to miniprepping the biobrick version of KerUS we created, the results obtained using the equipment and miniprep kit at our school was less successful. We hypothesize this is largely because we were unable to obtain highly concentrated alcohols to add to the kit, and possibly because of the DNA shearing as we use a dremelfuge (dremel tool centrifuge created in our first year of iGEM on 2014). To solve these problems a different miniprep protocol was developed.

The results obtained using this protocol are unclear. There seems to be a large smear of DNA on the gel as opposed to two clear bands (two because of the supercoiled and relaxed conformation of the plasmids). This once again seems to be due to shearing of DNA with the centrifugation. After realizing the error with the synthesis we realize that the problem with the miniprep is just that the bacteria do not contain the plasmid. However, as seen in the picture below there does seem to be more DNA than in the samples prepared using the filter columns in the kit.

Considerations for future experiments: At the moment it is hard to tell whether the poor results are due to the protocol or because of unsuccessful creation of the KerUS biobrick. Therefore in the future, a miniprep will be conducted using an RFP culture to verify the effectiveness of the miniprep. However, compared to not seeing any DNA with samples prepared with the non-modified miniprep this already seems more promising. The steps used to create the biobrick version of KerUS will also be repeated.


Future Plans:
Future plans include characterizing the properly created the new biobrick parts. AS well as re-creating the KerUS biobrick and conducting the different experiments with more replicates. If discovered to be truly toxic to E.coli, cell free protein synthesis could be another avenue for the team to explore, but at the moment proves to be too expensive and potentially unecessary.

Contact us at:
https://www.facebook.com/OLeSsence/
@igem_canmore
larvisais@redeemer.ab.ca