Team:HKU-Hong Kong/Lab Diaries

From 2011.igem.org

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|-
|-
|style="width:900px;"|'''Week 1'''
|style="width:900px;"|'''Week 1'''
 +
Transformation of reporter DNA (pEGFP-loxp-km-loxp) into DH10B (non-virulent strain E. coli) with antibiotic resistance (Chloramphenicol – Cm)
 +
|-
 +
|style="width:900px;"|'''Week 2'''
<OL>
<OL>
-
<LI>Transformation of reporter DNA (pEGFP-loxp-km-loxp) into DH10B (non-virulent strain E. coli) with antibiotic resistance (Chloramphenicol – Cm). To test the efficiency of transformation at different concentration, 3ug/uL, 30ug/uL and 300ug/uL bacterial cell culture were used. 100uL of each sample was used to do spread plates and incubated at 37C overnight. It was found that 3ug/uL of DNA was already enough to have high transformation efficiency of our DH10B competent cells. However, the colonies were very small. This might be because the bacteria grow very slowly. 5 colonies were inoculated from the plate which had been spread with 3ug/uL reporter DNA transformed DH10B separately into 3mL LB broth with 3uL Amp and incubated at 37C for several hours (from ~09:30 to ~14:30) to see if it could really grow in growth medium. Results were failed because DH10B was Cm resistant but not Amp resistant. 5 colonies were inoculated again from the plate which had been spread with 3ug/uL reporter DNA transformed DH10B separately. But this time into 3mL LB broth with 3uL Cm and incubated at 37C overnight. Results were failed again, no turbidity in all the tubes. Perhaps it was because the bacteria grow too slowly. So we put it on the rotary for longer time. There was still no results, no turbidity. It might be because the colonies are so small that when we picked the colonies for inoculation, the bacterial cells could not be picked up by the tips and still stuck to the agar plate; or, the bacterial cells get stuck to the tips without really getting into the LB broth. Inoculation was carried out again as well as plasmid extraction (using mini-prep).</LI>
+
<LI>tetO2 -1 (DNA binding site)
-
</OL>
+
<OL>
 +
<LI>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.
 +
<LI>pEGFP-loxp-km-loxp-tetO2-1 was then transformed into DH10B to greatly amplify the product by using bacterial cells.
 +
</OL>
 +
<LI>tetO2 -2 (DNA biding site)
 +
<OL>
 +
<LI>Reverse PCR was used to insert tetO2 -2 into pEGFP-loxp-km-loxp.
 +
<LI>2uL of 10-fold diluted pEGFP-loxp-km-loxp- tetO2 -2 was transformed into DH10B to greatly amplify the product by using bacterial cells.
 +
</OL>
 +
<LI>tetO2 – 3 (DNA binding site)
 +
<OL>
 +
<LI>Reverse PCR was used to insert tetO2 – 3 into pEGFP-loxp-km-loxp.
 +
<LI>2uL of 10-fold diluted pEGFP-loxp-km-loxp- tetO2 – 3 was transformed into DH10B to greatly amplify the product by using bacterial cells.
 +
</OL>
 +
|-
 +
|style="width:900px;"|'''Week 3'''
 +
<OL>
 +
<LI>Overlap PCR (involving 3 steps) was carried out to produce the fusion protein [tetR-HNS (any length)].
 +
<OL>
 +
<LI>PCR was carried out separately for 2 genes, tetR and HNS (full length, in this case).
 +
  <OL>
 +
  <LI>Primers used were as below:
 +
  <LI>tetR: [forward] R-H-out-F; [reverse] R-H-tetR-R
 +
  <LI>HNS (FL): [forward] R-H-tetR-F-out; [reverse] R-H(FL)-2-R
 +
<div ALIGN=CENTER>
 +
{| style="width:254px;background:#99EE63;text-align:center;font-family: georgia, helvetica, arial, sans-serif;color:#000000;margin- top:5px;padding: 2px;" cellspacing="5";
 +
|-
 +
|[[Image:KAREN_Fusion_protein_gene.png|250px]]
 +
|-
 +
|Fusion protein genes produced separately using PCR
 +
|}
 +
</div>
 +
  </OL>
 +
<LI>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.
 +
<LI>PCR was used to further amplify the product from step 2 (fusion protein gene).
 +
</OL>
 +
<LI>PCR was used to obtain HNS(FL) at 5 different temperatures to test which temperature is optimal for annealing.
 +
<OL>
 +
<LI>Annealing phase at 5 different temperatures:
 +
  <OL>
 +
  <LI>60.7C
 +
  <LI>62.2C
 +
  <LI>63.8C
 +
  <LI>65.4C
 +
  <LI>66.7C (less sample)
 +
  </OL>
 +
<LI>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.
 +
 
 +
<div ALIGN=CENTER>
 +
{| style="width:254px;background:#99EE63;text-align:center;font-family: georgia, helvetica, arial, sans-serif;color:#000000;margin- top:5px;padding: 2px;" cellspacing="5";
 +
|-
 +
|[[Image:KAREN_fusion_ptn_2.jpg|250px]]
 +
|-
 +
|Fusion protein genes annealed at 5 different temperatures
 +
|}
 +
</div>
 +
|-
 +
|style="width:900px;"|'''Week 4-5'''
 +
tetO2 – 0 and tetO2 – 4
 +
<OL>
 +
<LI>Reverse PCR was used to insert tetO2 -0 and tetO2 – 4 into pEGFP-loxp-km-loxp.
 +
<LI>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.
 +
|-
 +
|style="width:900px;"|'''Week 6'''
 +
Overlap PCR was carried out to produce fusion protein gene
 +
<OL>
 +
<LI>HNS::tetR
 +
  <OL>
 +
  <LI>HNS (1-46)
 +
  <LI>HNS (1-83)
 +
  <LI>HNS (1-90)
 +
  <LI>HNS (FL)
 +
  </OL>
 +
<LI>tetR::HNS
 +
  <OL>
 +
  <LI>HNS (2-46)
 +
  <LI>HNS (2-83)
 +
  <LI>HNS (FL)
 +
  </OL>
 +
</OL>
 +
|-
 +
|style="width:900px;"|'''Week 7'''
 +
Overlap PCR was carried out to produce fusion protein gene tetR::HNS (2-90)
 +
<OL>
 +
<LI>The sticky ends of the enzyme products was transformed into blunt ends using PCR.
 +
|-
 +
|style="width:900px;"|'''Week 8'''
 +
<OL>
 +
<LI>pROT-HNS (for BioBricks) was produced
 +
<LI>Double enzyme digestion was carried out to digest the fusion protein genes and ligate them to pBS1C3 (plasmid backbone)
 +
<OL>
 +
<LI>3 samples were transformed
 +
  <OL>
 +
  <LI>pSB1C3-tetR::HNS (2-83)
 +
  <LI>pSB1C3-tetR::HNS (2-90)
 +
  <LI>pSB1C3-tetR::HNS (FL)
 +
  </OL>
 +
</OL>
 +
<LI>Enzyme digestion was carried out for the extracted and purified plasmid tetO2-0 and tetO2-3
 +
<OL>
 +
<LI>0.5uL of CIP alkaline phosphatase was added to prevent self-ligation to each enzyme digest reaction mixture.
 +
|-
 +
|style="width:900px;"|'''Week 9'''
 +
<OL>
 +
<LI>tetO2 – 0 and tetO2 – 3 were ligated to form tetO2 – 0 – 3.
 +
<OL>
 +
<LI>tetO2 – 0 – 3 was transformed to DH10b competent cells.
 +
</OL>
 +
<LI>Production of tetO2 – 0 –sfGFP
 +
 
 +
<div ALIGN=CENTER>
 +
{| style="width:254px;background:#99EE63;text-align:center;font-family: georgia, helvetica, arial, sans-serif;color:#000000;margin- top:5px;padding: 2px;" cellspacing="5";
 +
|-
 +
|[[Image:TetO2-0-sfGFP.jpg|250px]]
 +
|-
 +
|tetO2-0-sfGFP
 +
|}
 +
</div>
 +
|-
 +
|style="width:900px;"|'''Week 10'''
 +
<OL>
 +
<LI>EcoRI and PstI enzyme cut sites were added to both ends of HNS using PCR (for BioBricks construction).
 +
<LI>Production of
 +
<OL>
 +
<LI>J23103RH
 +
<LI>J23106RH; J23106RHH
 +
<LI>J23116RH; J23116RHH
 +
<LI>pET28a
 +
<LI>pSB1C3-tdRema
 +
</OL>
 +
<LI>Checking fluorescence intensity of sfGFP and EGFP
 +
<OL>
 +
<LI>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.
 +
<LI>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.
 +
<LI>Fluorescence intensity were checked at OD600 using spectrophotometer
 +
</OL>
 +
|-
 +
|style="width:900px;"|'''Week 11'''
 +
<OL>
 +
<LI>Production of
 +
<OL>
 +
<LI>J23103R
 +
<LI>J23106R
 +
<LI>J23109R
 +
<LI>J23116R
 +
<LI>J23103RH
 +
<LI>J23106RH
 +
<LI>J23109RH
 +
<LI>J23116RH
 +
<LI>J23103RHH
 +
<LI>J23106RHH
 +
<LI>J23109RHH
 +
<LI>pSB1C3-tetR::HNS (2-83)
 +
<LI>pSB1C3-tetR::HNS (2-90)
 +
<LI>pSB1C3-tetR::HNS (FL)
 +
<LI>pSB1C3-HNS
 +
<LI>Electrophoresis was carried out to confirm the success of extraction) (except J23116RH)
 +
 
 +
<div ALIGN=CENTER>
 +
{| style="width:254px;background:#99EE63;text-align:center;font-family: georgia, helvetica, arial, sans-serif;color:#000000;margin- top:5px;padding: 2px;" cellspacing="5";
 +
|-
 +
|[[Image:Week_11.jpg|250px]]
 +
|-
 +
|Confirmation of successful extraction
 +
|}
 +
</div>
 +
 
 +
<div ALIGN=CENTER>
 +
{| style="width:254px;background:#99EE63;text-align:center;font-family: georgia, helvetica, arial, sans-serif;color:#000000;margin- top:5px;padding: 2px;" cellspacing="5";
 +
|-
 +
|[[Image:Week_11_(2).jpg|250px]]
 +
|-
 +
|Confirmation of successful extraction
 +
|}
 +
</div>
 +
</OL>
 +
<LI>Purification of NdeI digested pET28a
 +
|-
 +
|style="width:900px;"|'''Week 12'''
 +
<OL>
 +
<LI>Production of
 +
<OL>
 +
<LI>Bio-83
 +
<LI>Bio-90
 +
<LI>Bio-FL
 +
<LI>Bio-HNS
 +
</OL>
 +
<LI>Transformation to competent cells using electroporation
 +
<OL>
 +
<LI>tetO2 – 0
 +
<LI>tetO2 – 1
 +
<LI>sfGFP
 +
|-
 +
|style="width:900px;"|'''Week 13'''
 +
<OL>
 +
<LI>Production of
 +
<OL>
 +
<LI>Bio-HNS
 +
<LI>Bio-HNS
 +
<LI>pET28a-tetR
 +
<LI>pET29a-HNS
 +
<LI>pET28a-tetR::HNS (2-90)
 +
</OL>
 +
<LI>Transformation of
 +
<OL>
 +
<LI>J23106RH
 +
<LI>J23103R
 +
<LI>J23109RHH
 +
<LI>J23106R
 +
<LI>J23109R
 +
<LI>J23103RH
 +
<LI>J23116R
 +
|-
 +
|style="width:900px;"|'''Week 14'''
 +
<OL>
 +
<LI>Production of J23116-tetR::HNS (2-90)
 +
<OL>
 +
<LI>Colony PCR was used to confirm the success of production
 +
</OL>
 +
<LI>Fluorescence of our samples were tested [in M1655 (with tetO-0, tetO-1 or tetO-0-sfGFP)]
 +
<OL>
 +
<LI>J23109R
 +
<LI>J23109RH
 +
<LI>J23109RHH
 +
<LI>J23116R
 +
<LI>J23116RHH
 +
</OL>
 +
<LI>Check for the OD488 to OD510 (green fluorescence); OD600 (cell concentration in the solution)
 +
<OL>
 +
<LI>With 1 set for CTC
 +
  <OL>
 +
  <LI>Procedures
 +
  <OL>
 +
  <LI>A colony of the above samples was incubated in 3mL LB broth with antibiotics overnight.
 +
  <LI>1:50 diluted in M9 solution (10mL) (for CTC set: add CTC 20ug/mL).
 +
  <LI>Samples were then incubated at 37C for 6 hours in dark.
 +
  <LI>Each sample was then concentrated to 200uL.
 +
  <LI>Samples were loaded into a 96-well plate to check for OD using spectrophotometer.

Latest revision as of 06:59, 5 October 2011

Lab Diaries
Week 1

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

Week 2
  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.
Week 3
  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
        KAREN Fusion protein gene.png
        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.
      KAREN fusion ptn 2.jpg
      Fusion protein genes annealed at 5 different temperatures
Week 4-5

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.
Week 6

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)
Week 7

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.
Week 8
  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.
Week 9
  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.jpg
    tetO2-0-sfGFP
Week 10
  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
Week 11
  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)
      Week 11.jpg
      Confirmation of successful extraction
      Week 11 (2).jpg
      Confirmation of successful extraction
  2. Purification of NdeI digested pET28a
Week 12
  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
Week 13
  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
Week 14
  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.