Team:KUAS Korea/Notebook

Notebook

Notebook

Week 1.

  1. We researched several gastrointesintal diseases and its causes.
  2. Additionally, we researched existing diagnostic methods used in detecting these diseases.

Week 2.

  1. We looked for gut microbiome distribution in the gut and bacterias that are currently verified and utilized in probiotics.
  2. Also, we chose heme, pH, bile salt as our target indicator, which could detect the diseases we previously researched on the first week.

Week 3.

    We designed a detector device based on analyzation of prior research about sensing heme, pH, and bile salt.

Week 4.

  1. We received Lactobacillus plantarum L67 from professor Se Jong Oh of Chunnam University, and cultured them in MRS media.
  2. We collected single colony from them and cultured it.
  3. We then cultured these bacteria in culture medium that contain ampicillin, chloramphenicol, tetracycline, gentamycin, rifampicin, streptomycin, kanamycin, and tested antibiotic resistance.

Week 5.

  1. 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.
  2. We compared the test result and verified antibiotic resistance data of L. plantarum, and checked if the right bacteria was cultured.
  3. We researched on transformation protocol of Lactobacillus transformation and referenced protocol of Eppendorf.

Week 6.

  1. We selected the gram positive-E. coli shuttle vector for the transformation of the Lactobacillus Plantarum L67
  2. 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.
  3. We measured the concentration and the checked the size of the outcome using gel electrophoresis.

Week 7.

  1. Lactobacillus Plantarum L67 Electroporation competent cell was produced. (Eppendorf protocol was referred)
  2. We tried to transform previously amplified vector to the competent cell at various voltages and amounts of vector.
  3. Result was checked using the selective culture.

Week 8.

    After the failure of transformation on the 7th week, we re-constructed competent cell and carried out another experiment.

Week 9.

  1. 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.
  2. Our professor revised the previously designed detecting device.
  3. We ordered forward and reverse primer for PCR of hssR and hssS from the IDT.
  4. We also ordered sequences of hrtR promoter and HrtR protein attached with iGEM prefix and suffix.

Week 10.

    We requested for Bacillus cereus ATCC from Korean Collection for Type Culture(KCTC) 14579.

Week 11.

  1. We cultured freeze-drying B. cereus from KCTC on Nutrient Agar broth KCTC and got a single colony.
  2. We ordered psH71 replicon of Lactic Acid Bacteria from IDT.

Week 12.

  1. gBlock and primer which we ordered from IDT at week 9 have arrived.
  2. gDNA of B. cereus was extracted.
  3. we processed the PCR, using gDNA and primer but the band corresponding to HssRS was not detected on gel electrophoresis.

Week 13.

  1. Retried PCR but failed.
  2. We requested BBa_K1033282 to the iGEM Headquarter.

Week 14.

  1. We selected three preexisting part from the iGEM distribuition kit for Characterization.
  2. BBa_K1033282 arrived from iGEM HQ.
  3. We cultured E. coli Dh5alpha from our cell stock, and made heat-shock competent cell.
  4. We cloned hrtR gBlock in the pSB1C3 vector.

Week 15.

  1. We transformed four existing part in the DH5 alpha and confirmed that two parts, BBa_K1033282, BBa_K1696009 had succeeded transforming.
  2. The cloning of gBlock also succeeded.

Week 16.

  1. Seed culture of the DH5alpha which has BBa_K1033282(amilCP), was done.
  2. OD588 was measured at 20 min interval on preparatory experiments.
  3. Although pSB1C3 was inserted, additional TA cloning was done since it was hard to sort colonies not having the gBlock.

Week 17.

  1. We cultured BBa_K1033282 inserted DH5alpha and measured absorbance of OD588(maximum absorbance) and OD800(cell concentration) in 30 minute interval.
  2. 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.
  3. We uploaded these graphs and photos on the wiki main page of BBa_K1033282.
  4. We acquired pSB1C3 with HrtR gblock inserted as a result of TA cloning.
  5. We digested this by restriction enzyme again, and confirmed the size of the digested DNA fragment.
  6. We submitted pSB1C3-hrtR to the iGEM HQ.