Difference between revisions of "Team:KUAS Korea/Notebook"

Week 1.

1. We researched several gastrointesintal diseases and its cause.
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.

Week 13.

Week 14.

Week 15.

Week 16.

1. We studied a paper that fixation of diaphorase onto electrodes.
2. But experiments were too complicated, so we gave up.

Week 17, 18

1. We did an MFC validation with Shewanella, phosphate buffer saline(PBS), and galactose.
2. We did same experiment 3 times.

Week 19.

1. I changed the mulitimeter's circuit to test 5 fuel cell at the same time.
2. We did EFC test with 3 sample, E. coli(Diaphorase expressing), yeast, and E. coli(Diaphorase expressing) + yeast.
3. All cells was lysed by ultrasonication.

Week 20.

1. We did similar experiment with last week, but sample was treated with centrifuge.
2. We thought that the agarase in anolyte can react with agar in salt bridge, so we paste cellophane membrane at anode part of salt bridge. Cellophane membrane was fixed by 15mL Falcon tube's cap.
3. Part cloning was done

Week 21.

1. I made 6 new device. The parts with pasted with glue gun was melted during autoclaving, so I changed it with waterproof silicon adhesive.
2. We did a MFC validation with Shewanella, E. coli BW25113, and a control group(No cells).
3. We did the experiment 3 times.

Week 22.

1. We did an EFC validation this week.
2. Transformed E. coli treated with arabinose or IPTG induction depending on its function.
3. We did the experiment 3 times.

Week 23.

1. The EFC validation graph looks strange.
2. Our professor told us that the amount of NADH might be too small.
3. So we did the experiment again with adding some NADH.

Week 24.

1. We created the whole EMFC system, and we measure the voltage. External resistance was 1,000 Ohm.
2. We draw the graph of EMFC, and compared with contrast.