Team:HKU-Hong Kong/Lab Diaries

Transformation of reporter DNA (pEGFP-loxp-km-loxp) into DH10B (non-virulent strain E. coli) with antibiotic resistance (Chloramphenicol – Cm)

1. tetO2 -1 (DNA binding site)
   1. Reverse PCR was used to insert tetO2 -1 into pEGFP-loxp-km-loxp (template DNA) by using forward and reverse primers which are with the tetO2 -1. By using reverse PCR, we can insert the tetO2 -1 site into the pEGFP while the plasmid produced is still in double-stranded circular form.
   2. pEGFP-loxp-km-loxp-tetO2-1 was then transformed into DH10B to greatly amplify the product by using bacterial cells.
2. tetO2 -2 (DNA biding site)
   1. Reverse PCR was used to insert tetO2 -2 into pEGFP-loxp-km-loxp.
   2. 2uL of 10-fold diluted pEGFP-loxp-km-loxp- tetO2 -2 was transformed into DH10B to greatly amplify the product by using bacterial cells.
3. tetO2 – 3 (DNA binding site)
   1. Reverse PCR was used to insert tetO2 – 3 into pEGFP-loxp-km-loxp.
   2. 2uL of 10-fold diluted pEGFP-loxp-km-loxp- tetO2 – 3 was transformed into DH10B to greatly amplify the product by using bacterial cells.

1. Overlap PCR (involving 3 steps) was carried out to produce the fusion protein [tetR-HNS (any length)].
   1. PCR was carried out separately for 2 genes, tetR and HNS (full length, in this case).
      1. Primers used were as below:
      2. tetR: [forward] R-H-out-F; [reverse] R-H-tetR-R
      3. HNS (FL): [forward] R-H-tetR-F-out; [reverse] R-H(FL)-2-R

         Fusion protein genes produced separately using PCR

   2. Another PCR was carried out using primers with linker to insert the linker DNA to the tetR and HNS (FL) for the fusion of the 2 genes.
   3. PCR was used to further amplify the product from step 2 (fusion protein gene).
2. PCR was used to obtain HNS(FL) at 5 different temperatures to test which temperature is optimal for annealing.
   1. Annealing phase at 5 different temperatures:
      1. 60.7C
      2. 62.2C
      3. 63.8C
      4. 65.4C
      5. 66.7C (less sample)
   2. Electrophoresis was carried to determine which temperature best suit the annealing phase. From the gel image, it was observed that a high concentration of products was resulted at annealing temperature between 60.7C and 62.2C.

      Fusion protein genes annealed at 5 different temperatures

tetO2 – 0 and tetO2 – 4

1. Reverse PCR was used to insert tetO2 -0 and tetO2 – 4 into pEGFP-loxp-km-loxp.
2. 2uL of pEGFP-loxp-km-loxp- tetO2 – 0 and pEGFP-loxp-km-loxp- tetO2 – 4 were transformed into DH10B separately to greatly amplify the product by using bacterial cells.

Overlap PCR was carried out to produce fusion protein gene

1. HNS::tetR
   1. HNS (1-46)
   2. HNS (1-83)
   3. HNS (1-90)
   4. HNS (FL)
2. tetR::HNS
   1. HNS (2-46)
   2. HNS (2-83)
   3. HNS (FL)

Overlap PCR was carried out to produce fusion protein gene tetR::HNS (2-90)

1. The sticky ends of the enzyme products was transformed into blunt ends using PCR.

1. pROT-HNS (for BioBricks) was produced
2. Double enzyme digestion was carried out to digest the fusion protein genes and ligate them to pBS1C3 (plasmid backbone)
   1. 3 samples were transformed
      1. pSB1C3-tetR::HNS (2-83)
      2. pSB1C3-tetR::HNS (2-90)
      3. pSB1C3-tetR::HNS (FL)
3. Enzyme digestion was carried out for the extracted and purified plasmid tetO2-0 and tetO2-3
   1. 0.5uL of CIP alkaline phosphatase was added to prevent self-ligation to each enzyme digest reaction mixture.

1. tetO2 – 0 and tetO2 – 3 were ligated to form tetO2 – 0 – 3.
   1. tetO2 – 0 – 3 was transformed to DH10b competent cells.
2. Production of tetO2 – 0 –sfGFP

   tetO2-0-sfGFP

1. EcoRI and PstI enzyme cut sites were added to both ends of HNS using PCR (for BioBricks construction).
2. Production of
   1. J23103RH
   2. J23106RH; J23106RHH
   3. J23116RH; J23116RHH
   4. pET28a
   5. pSB1C3-tdRema
3. Checking fluorescence intensity of sfGFP and EGFP
   1. 3 colonies from sfGFP streak plate and 1 colony from EGFP streak plate were picked. Each was inoculated into 0.5mL LB broth with 0.5uL Cm in 37C on rotary machine in warm room for around 3 hours.
   2. Samples were then diluted 1:50 in fresh 2mL LB broth with 2uL Cm in 37C on rotary machine in warm room for around 3 hours.
   3. Fluorescence intensity were checked at OD600 using spectrophotometer

1. Production of
   1. J23103R
   2. J23106R
   3. J23109R
   4. J23116R
   5. J23103RH
   6. J23106RH
   7. J23109RH
   8. J23116RH
   9. J23103RHH
   10. J23106RHH
   11. J23109RHH
   12. pSB1C3-tetR::HNS (2-83)
   13. pSB1C3-tetR::HNS (2-90)
   14. pSB1C3-tetR::HNS (FL)
   15. pSB1C3-HNS
   16. Electrophoresis was carried out to confirm the success of extraction) (except J23116RH)

       Confirmation of successful extraction

       Confirmation of successful extraction

2. Purification of NdeI digested pET28a

1. Production of
   1. Bio-83
   2. Bio-90
   3. Bio-FL
   4. Bio-HNS
2. Transformation to competent cells using electroporation
   1. tetO2 – 0
   2. tetO2 – 1
   3. sfGFP

1. Production of
   1. Bio-HNS
   2. Bio-HNS
   3. pET28a-tetR
   4. pET29a-HNS
   5. pET28a-tetR::HNS (2-90)
2. Transformation of
   1. J23106RH
   2. J23103R
   3. J23109RHH
   4. J23106R
   5. J23109R
   6. J23103RH
   7. J23116R

1. Production of J23116-tetR::HNS (2-90)
   1. Colony PCR was used to confirm the success of production
2. Fluorescence of our samples were tested [in M1655 (with tetO-0, tetO-1 or tetO-0-sfGFP)]
   1. J23109R
   2. J23109RH
   3. J23109RHH
   4. J23116R
   5. J23116RHH
3. Check for the OD488 to OD510 (green fluorescence); OD600 (cell concentration in the solution)
   1. With 1 set for CTC
      1. Procedures
         1. A colony of the above samples was incubated in 3mL LB broth with antibiotics overnight.
         2. 1:50 diluted in M9 solution (10mL) (for CTC set: add CTC 20ug/mL).
         3. Samples were then incubated at 37C for 6 hours in dark.
         4. Each sample was then concentrated to 200uL.
         5. Samples were loaded into a 96-well plate to check for OD using spectrophotometer.