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Revision as of 03:57, 10 October 2018

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iGEM Human Practice

Home Achievements Project Human-Practice Notebook Attributions

 

Collaboration

This year, we are very happy to collaborate with a collegiate iGEM team, CSMU_NCHU_Taiwan. We are both from Taichung City in Taiwan and the distance between us actually are very close, that is, not far from 5km.

In the meetup, CSMU_NCHU_Taiwan knew our project design and the problem to get from IDT the synthetic gene expression vector carrying glucose transporter genes of Salmonella. They advised that we can try to clone the gene from the bacteria and then assemble with the promoter. However, in safety issue, Salmonella is recognized as Biosafety Level 2 organism, they helped us to contact a lab PI, Dr. Cheng-Yang Huang, in Chung Shan Medical University who has handled the bacteria. Luckily, the genomic DNAs of Salmonella has been prepared for their previous research and kept some stored in the fridge. CSMU_NCHU_Taiwan amplified the gene fragments we want by PCR using our designed primers.

And in the other way, we did them a favor by subcloning BioBrick parts. We have good training in the skills of gene cloning learned from our instructor, Dr. Phil Chen. We have actually completed total at least 28 basic and composite BioBrick parts this year. And due to the limited remaining time of iGEM competition, CSMU_NCHU_Taiwan sought help from us to subclone parts from the synthetic genes made by IDT.

MINGDAO

We helped CSMU_NCHU_Taiwan subclone one basic part of “F420-Dependent Glucose-6-phosphate Dehydrogenase” and one composite part of “T7 promoter & Lac operator and RBS from PET-29a”. We took two tubes containing DNA powder made by IDT from CSMU_NCHU_Taiwan. We dissolved the DNA and re-transformed E. coli DH5α with them. Then the two plasmids extracted from the bacteria have been cut by XbaI and PstI as inserts, and so did the pSB1C3 as a vector (Fig 1.). E. coli DH5α competent cells were transformed with the ligation products. The transformed E. coli colonies were picked up and checked by colony PCR with VF2 and VR primers (Fig 2.). And one of these from each group were further checked with restriction enzymes of EcoRI and PstI (Fig 3.). Finally before giving back to CSMU_NCHUI_Taiwan, the plasmids were confirmed by DNA sequencing. In addition, we drew the plasmid map in VectorNTI format for them.

Fig 1. The plasmids were cut by XbaI and PstI. Lane 1: pSB1C3 (2044 bp+ 1095 bp). Lane 2: F420-Dependent Glucose-6-phosphate Dehydrogenase/pUCIDT (2769 bp+ 1032 bp). Lane 3: T7 promoter & Lac operator and RBS from PET-29a/pIDTSMART(Amp) (2073 bp+109 bp)

Fig 2. The results of colony PCR with VF2 and VR primers on the transformed colonies. Left gel: F420-Dependent Glucose-6-phosphate Dehydrogenase/pSB1C3 (1320 bp); Right gel: T7 promoter & Lac operator and RBS from PET-29a/pSB1C3 (397 bp)
Fig 3. Restriction enzyme digestion check by EcoRI and PstI for plasmids extracted from the transformed E. coli which have been confirmed by colony PCR

Fig 4. The map of F420-Dependent Glucose-6-phosphate Dehydrogenase/pSB1C3 in VectorNTI format

Fig 5. The map of T7 promoter & Lac operator and RBS from PET-29a/pSB1C3 in VectorNTI format

CSMU_NCHU_Taiwan

CSMU_NCHU_Taiwan helped us amplify the glucose transporter genes from gDNA of Salmonella typhimurium LT2. We designed the specific primers with or without RBS (B0034) sequence which are targeting to three transporter genes, crr, ptsG and STM1128, respectively. CSMU_NCHU_Taiwan took the primer sets and performed PCR for us. And they also did a positive control to check the integrity and identity of genomic DNA of Salmonella typhimurium LT2. The PCR-amplified DNA fragments were shown in the DNA agarose gel in Fig 6.

Fig 6. The amplified DNA fragments of glucose transporter genes by PCR from gDNA of Salmonella typhimurium LT2. Lanes 1. crr (510 bp); 2. RBS-crr (528 bp); 3. ptsG (1434 bp); 4. RBS-ptsG (1452bp); 5. 1128 (1497 bp); 6. RBS-1128 (1515 bp); 7. Positive control: STM3098 (423 bp), Appl Environ Microbiol. 2006.

  • - REFERENCE -
  • 1.     Comparison of Salmonella enterica Serovar Typhimurium LT2 and Non-LT2 Salmonella Genomic Sequences, and Genotyping of Salmonellae by Using PCR. Appl Environ Microbiol. 2006;72(9): 6142–6151.
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