- We researched several gastrointesintal diseases and its causes.
- Additionally, we researched existing diagnostic methods used in detecting these diseases.
- We looked for gut microbiome distribution in the gut and bacterias that are currently verified and utilized in probiotics.
- Also, we chose heme, pH, bile salt as our target indicator, which could detect the diseases we previously researched on the first week.
We designed a detector device based on analyzation of prior research about sensing heme, pH, and bile salt.
- We received Lactobacillus plantarum L67 from professor Se Jong Oh of Chunnam University, and cultured them in MRS media.
- We collected single colony from them and cultured it.
- We then cultured these bacteria in culture medium that contain ampicillin, chloramphenicol, tetracycline, gentamycin, rifampicin, streptomycin, kanamycin, and tested antibiotic resistance.
- Our Professor gave us a feedback on the experiment conducted in week 4. Based on the feedback, we revised the problematic part and conducted the experiment again.
- We compared the test result and verified antibiotic resistance data of L. plantarum, and checked if the right bacteria was cultured.
- We researched on transformation protocol of Lactobacillus transformation and referenced protocol of Eppendorf.
- We selected the gram positive-E. coli shuttle vector for the transformation of the Lactobacillus Plantarum L67
- After taking the shuttle vector out from stock, heat-shock transformation to E. coli BW25113 was processed to amplify the vector. vector was extracted by mini-prep method.
- We measured the concentration and the checked the size of the outcome using gel electrophoresis.
- Lactobacillus Plantarum L67 Electroporation competent cell was produced. (Eppendorf protocol was referred)
- We tried to transform previously amplified vector to the competent cell at various voltages and amounts of vector.
- Result was checked using the selective culture.
After the failure of transformation on the 7th week, we re-constructed competent cell and carried out another experiment.
- After the re-failure of transformation on the 8th week, we found out another protocol and constructed a new competent cell and performed an experiment once again.
- Our professor revised the previously designed detecting device.
- We ordered forward and reverse primer for PCR of hssR and hssS from the IDT.
- We also ordered sequences of hrtR promoter and HrtR protein attached with iGEM prefix and suffix.
We requested for Bacillus cereus ATCC from Korean Collection for Type Culture(KCTC) 14579.
- We cultured freeze-drying B. cereus from KCTC on Nutrient Agar broth KCTC and got a single colony.
- We ordered psH71 replicon of Lactic Acid Bacteria from IDT.
- gBlock and primer which we ordered from IDT at week 9 have arrived.
- gDNA of B. cereus was extracted.
- we processed the PCR, using gDNA and primer but the band corresponding to HssRS was not detected on gel electrophoresis.
- Retried PCR but failed.
- We requested BBa_K1033282 to the iGEM Headquarter.
- We selected three preexisting part from the iGEM distribuition kit for Characterization.
- BBa_K1033282 arrived from iGEM HQ.
- We cultured E. coli Dh5alpha from our cell stock, and made heat-shock competent cell.
- We cloned hrtR gBlock in the pSB1C3 vector.
- We transformed four existing part in the DH5 alpha and confirmed that two parts, BBa_K1033282, BBa_K1696009 had succeeded transforming.
- The cloning of gBlock also succeeded.
- Seed culture of the DH5alpha which has BBa_K1033282(amilCP), was done.
- OD588 was measured at 20 min interval on preparatory experiments.
- Although pSB1C3 was inserted, additional TA cloning was done since it was hard to sort colonies not having the gBlock.
- We cultured BBa_K1033282 inserted DH5alpha and measured absorbance of OD588(maximum absorbance) and OD800(cell concentration) in 30 minute interval.
- We drew graphs showing the change of OD588 in accordance to time, the change of OD588/OD800 ratio to time, and change of OD588 in accordance to OD800 value.
- We uploaded these graphs and photos on the wiki main page of BBa_K1033282.
- We acquired pSB1C3 with HrtR gblock inserted as a result of TA cloning.
- We digested this by restriction enzyme again, and confirmed the size of the digested DNA fragment.
- We submitted pSB1C3-hrtR to the iGEM HQ.