Unless otherwise stated all experiments and lab work was conducted in our school Biotechnology lab. Permission was granted to us to work on campus under supervision from Monday to Friday, until 4:00pm during July of our summer holidays. After the summer, lab work again commenced at the beginning of the new semester (August 20th) as part of our Extra Curricular activity BioBuilder club under teacher supervision.
Week 1 – July 2nd to 6th
Lab safety training. steak plate method practice. cell culturing practice using TOP10 cells, aseptic technique. Autoclave safety training. And correct disposal of bio-waste training.
Week 2 – July 9th to 13th
Making LB agar plates with Kanamycin antibiotic and stock LB liquid agar for storage. Kanamycin stock solution (x1000) made. Stored at 4oC.
Week 3 – July 16th to 20th
Interlab studies. Transformation of plasmids into DH5 alpha cells and incubated for 16hrs @ 37oC. (Our Lab). Cell measurement data using plate reader (ECUST lab).
Week 4 – July 23rd to 27th
Transformation protocol practice, digestion and ligation protocol practice. Antibiotic test using disc diffusion assay method.
Week 5 - Aug 20th to 24th
Transformation of pET 30a plasmid with our designed biosynthetic gene cluster into with BL21 DE3 competent cells via Heat shock method. Plating with transformed cells onto Kanamycin antibiotic plates with negative control (untransformed cells to ensure antibiotic works) and incubated overnight @ 37oC. Cell counts.
Week 6 – Aug 27th to Aug 31st
Creating glycerol stocks solution (20% glycerol). Creating glycerol stocks of transformed cells (1:1 ratio). Stored at -80oC in ECUST lab. Cell growth curves developed.
Week 7 – Sept 3rd to 8th
Making CAS agar plates (needed 3 attempts as first batch yielded plates with a slight green color, inaccurate pH control was blamed. Batches 2 & 3 showed improvements and a more blue color was observed. Due to lack of concentrated HCl for glassware washing these plates were decided to be adequate). Dry lab: PCR amplification primer design.
Week 8 – Sept 10th to 14th
Induction of gene cluster in cells using IPTG. Different concentrations of IPTG were used to induce the gene cluster (0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM, plus a control of cells w/o IPTG) when cell growth was at OD600 = 0.6. IPTG was added to cell cultures and induced for 3 hours at 37oC. Cell cultures were centrifuged and cell free supernatant was used for a CAS plate assays using disc diffusion method (results were difficult to interpret).
Week 9 – September 17th to 21st
Repetition of induction of gene cluster experiments but for only IPTG concentrations of 0.4mM, 0.8mM based on disc diffusion plate assays observations. CAS assay plate assay was conducted by creating a 5mm plug in plates and filling plug with 30µl of cell free supernatant. Preparation of samples for LC-MS analysis (outside laboratory) – cell free supernatant of samples was frozen using dry ice and shipped immediately to laboratory.
Week 10 - Sept 24th to 28th
CAS liquid solution created and CAS liquid assay completed. Absorbance measurements @ 630nm Collaboration - sharing protocol with ECUST and providing CAS agar plates to team ECUST. Analysis of absorbance data. Calculation/Estimation of siderophore detection in samples.
Week 11- October 2nd to 6th
PCR amplification of targets. PCR purification and Digestion, Gel analysis of DNA length. Ligation of gene of interest to plasmid pSC13. Documentation of BioBricks.
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