Team:OLS Canmore/Experiments

• HOME
• TEAM
  Team Attributions
• PROJECT
  Description Design Experiments Proof of Concept Demonstrate Results Notebook
• PARTS
  Parts Basic Parts Composite Parts
• SAFETY
• PRACTICES
  Human Practices Integrated Practices Engagement Collaborations
• AWARDS
  Silver Gold

EXPERIMENTS

General
All general protocols including those for agarose gel electrophoresis, miniprep, restriction digest, making LB broth, and more can be found in the Geekstarter and AITF guidebook for high school iGEM. It can be found here. The protocols are found on pages 121 to 142.

Making Skim Milk plates
The skim milk plates are used to show enzyme activity. As keratinase is an enzyme with a broad substrate specificity, zones of clearing should be seen around cultures grown or supernatant of cell lysis poured on skim milk due to keratinase hydrolyzing the casein in the skim milk.

To make skim milk plates, 125mL of agar was prepared according to methods published in the AITF guidebook (p. 135-136) linked above. While autoclaving the 125mL LB agar for 25 minutes, 5g of skim milk powder was dissolved into 125mL distilled water. As the agar cooled the dissolved milk solution was autoclaved for 10 minutes. 2.5µL of Ampicillin was added to he LB agar, then the skim milk solution was poured in and mixed. Ampicillin was used as the assays we conducted were done with the non-biobrick versions of the parts, but the appropriate concentration of chloramphenicol can be substituted. Lastly, the plates are the poured and they should look opaque with no solids.

Cell Lysis
Because of the issues of other groups experienced with the expression of keratinase in E.coli, and the largely negative results of the semi-quantitative hair degradation assay we were curious to see if the problem was that keratinase is not being secreted, is secreted only at low quantities, or the gene is not being expressed properly. For this we designed a cell lysis protocol to lyse the cells without denaturing keratinase. The lysis allowed us to plate the supernatant on skim milk plates and observe any keratinolytic activity due to keratinase that was expressed but not properly secreted by the cells.

For lysing the cells, two overnight colonies of KerUS and a control of two JM109 were grown in LB broth. These cultures were diluted 100x in 5mL LB broth for a total of 8 tubes. 5µL ampicillin was added to cultures containing kerUS. Half the tubes also received 50µL of IPTG (100mM), whereas the other half received 50µL distilled water. The cultures were the incubated for 3.5 hours at 37°C. After incubation, 1.5mL of each of the kerUS cultures and JM109 cultures were centrifuged for 2 minutes at 7,000 RPM. This step was repeated twice to obtain a larger cell pellet.
To make the lysis buffer, 3.03g of Tris was stirred into 500mL demineralized and deionized water. This was refrigerated for an hour, and then 0.77g of DTT was added, as well as 0.5g of lysozyme.

After adding 60µL of the lysis buffer to each cell pellet, the tubes were shaken in the fridge for 30 minutes. The tubes were frozen in liquid nitrogen and thawed in a 40-42°C water bath; this was repeated twice. The tubes were centrifuged at 7,000 RPM for 4 minutes.
60µL of lysis buffer was added for a second time, and the tubes were spun again for 2 minutes at 7,000 RPM. The supernatants were taken off of the pellets for use in the skim milk plate assay. Link to the results.

Skim Milk plate assay
The skim milk plates are used to show enzyme activity. Cultures and supernatant containing keratinase should show zones of clearing as the enzyme would degrade the protein in the milk.

For plating of lysis supernatant:
10µL of each supernatant from each tube, obtained from the lysis protocol described above, was pipetted onto a skim milk plate in separate corners (four samples per plate). The plate(s) were kept at room temperature for four days.

For plating of cell culture:
10µL of cell culture is pipetted into separate corners of the plate (four samples per plate). The plate(s) were kept at room temperature for four days.
Note: the culture does not need to be induced with IPTG, despite using a plasmid regulated with a LacI promoter, because the lactose serves as a mimic.

In plating both the supernatant and cell culture it is important not to disturb the plate and to keep them on even surfaces so that the samples do not spread. Link to the results.

Semi-quantitative Hair Degradation assay
The semi-quantitative hair degradation assay was conducted to observe any potential enzyme activity on relatively large amounts of hair.

Using sterile LB broth, cultures of E.coli containing the non-biobrick kerA plasmid and non-biobrick kerUS plasmid, and without any plasmid were grown overnight at 37°C. Ampicillin was added to the cultures containing the kerA and kerUS plasmids for a final concentration 100µg ampicillin/mL. After 12 hours of incubation, the OD600 of each culture was measured before adding IPTG to the media with a final concentration of 1mM.

Human hair was dried at 65°C for 15 minutes until very dry. The hair was then weighed and distributed in 0.05g aliquots of loose hair to each of the cultures. The hair-containing cultures were then placed in the incubator at 37°C with gentle shaking.

After 120 hours of incubation, all tubes were centrifuged at 3,000 RPM for 5 minutes to cause hair to pellet, and the supernatant was discarded. After rinsing the remaining hair pellet in 10% bleach, they were dried overnight at 37°C. The dry hair was weighed to determine the final mass of hair. Link to the results.

Modified mini-prep
A miniprep is essential to isolate the plasmid in order to submit the biobricks to the iGEM registry. Unfortunately we experienced many troubles with our minipreps and it is one of the last steps we have to complete before submitting the parts. After all this trouble using our miniprep kit with filter columns as we could not procure the right concentration alcohols, we adjusted the protocol in an attempt to isolate plasmid DNA.

We use an Omega bio-tek E.Z.N.A. Plasmid DNA mini kit. The steps outlined in the protocol provided by Omega bio-tek with the kit are followed up until transferring the supernatant to the silica gel columns. Instead the 500µL supernatant is moved to new eppendorf tubes. Then 650µL of 99% isopropanol is added. The solution is inverted 12 times and incubated at room temperature for 10 minutes. The tubes are the spun at maximum speed for 10 minutes. The supernatant is gently poured off, while being sure not to disturb the glassy white pellet at the bottom of the tube.
500µL of ice cold 70% ethanol is added into the tube, and spun for 1 minute before pouring the liquid off. A white pellet should be seen at the bottom of the tube. The pellets are left to dry for 10 minutes to ensure all the ethanol has evaporated. Finally, 30-50µL of water is used to gently resuspend the pellet of DNA. Link to the results.

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