1. Designing the Heme sensing device
We have looked for the precedent researches on heme resistance of gut microbiome, we have decided to select the external detector system using HssS & HssR protein and the internal dectector utilizing HrtR protein. We designed a complete set comprised of promoter, sensing protein, and marker by inserting genetic marker instead of the efflux pump in the conventional mechanism.
2. Constructing the Heme sensing device
➀ In the case of HssS, HssR, we could find the matching sequence in Bacillus cereus ATCC 14579 by BLASTing the sequence that was mentioned in the former research. We received this bacteria from Korean Collection of Type Culture(KCTC), cultured them and collected a single colony. We extracted the gDNA of B.cereus, and added igem prefix, suffix respectively to the front and end of these HssR, HssS sequences. We also conducted PCR of gDNA template using this primer we designed.
➁ We modified HrtR and its promoter by adding iGEM prefix at the front, and suffix at the end. Moreover, we ordered from the IDT as a gBlock. We inserted the arrived gBlock utilizing the 3A assemble and TA cloning. We also screened the bacteria with ones that do not show the expression of RFP. We then cleaved the cloning vector using restriction enzymes and processed gel electrophoresis to check if the part was correct inserted.
3. Transformation of the Lactobacillus plantarum L67
We received L. plantarum L67 from the lab of Professor Se Jong Oh, Chonnam University. Then, we cultured the bacteria on MRS media and collected single colony. Then we tested antibiotic resistance to prove that we cultured the appropriate bacteria. For the selection of transformation vector of Lactobacillus, we have chosen the gram-positive shuttle vector that our lab already owned. We made electron competent cell of lactobacillus and attempted transformation in various conditions with different DNA amount and voltage. However, the transformation was unfortunately not successful, and we concluded that the reason was the malfunction of replicon in the Lactobacillus. So we additionally ordered the Lactic Acid Bacteria (LAB) replicon for BBa_K1033206.
4. Construction of the Screenable marker
When defining the presence of occult blood in our feces, we thought it would be difficult for users to recognize if we used general GFP, due to the low fluorescence. Therefore, we planned to use chromoprotein rather than fluorescent proteins like GFP. We found out that 2013 Uppsala team previously used Lactobacillus and chromoprotein in their project. So we received the part this team submitted from the iGEM HQ and tested expression.