Difference between revisions of "Part:BBa K4033004"
 
Line 20: Line 20:
 
<b><font size="3">Experimental approach</font></b>
 
<b><font size="3">Experimental approach</font></b>
  
 +
1. Pre growth
  
 +
1) Prepare 50mg / ml streptomycin mother liquor, filter and sterilize with filter membrane
 +
 +
2) Add 5ml LB liquid medium into 10ml centrifuge tube and 2ul streptomycin
 +
 +
3) Scrape some agar (including bacteria) along the puncture line with the inoculation ring, and extend the inoculation ring into the LB medium in the corresponding centrifuge tube to complete the inoculation
 +
 +
4) 37 ℃ 220 RPM overnight grow
 +
 +
2. Expression of protein
 +
 +
1） Add 10ml LB medium into 50ml centrifuge tube and 4ul chloramphenicol. Make two tubes for each plasmid, one of which is the control
 +
 +
2) 500ul of bacteria cultured overnight were added to 10ml LB medium and expanded at 37 ℃ and 220rpm for 2.5h
 +
 +
3） 0.1M IPTG 50ul (final concentration: 0.5mm) was added to the LB medium of the induction group of two plasmids, which was induced at 37 ℃ and 220rpm for 4H; The control group of the two plasmids did not add IPTG, and the parallel experiment was carried out
 +
 +
4） Suck 1ml bacterial solution from the four cultures, add it to 1.5ml EP tube, centrifuge at 12000rpm for 1min, discard the supernatant, add 50ul ddH2O to resuspend the bacteria, and then add 50ul SDS loading buffer
 +
 +
5） The mixed bacteria and loading buffer were heated at 100 ℃ for 10 min
 +
 +
6） Put the heated protein into - 20 ℃ for later use.
 +
 +
3. protein extraction
 +
 +
1) Draw 1ml bacterial solution from the cultures of the experimental group and the control group of each strain, add it to the 1.5ml EP tube, centrifuge at 12000rpm for 1min, discard the supernatant, add 50ul ddH2O to resuspend the bacteria, and then add 50ul SDS loading buffer
 +
 +
2) The mixed bacteria and loading buffer were heated at 100 ℃ for 10 min
 +
 +
3) Put the heated protein into - 20 ℃ for standby
 +
 +
4. SDS polyacrylamide gel Preparation for protein detection
 +
 +
1) Put the dried glass plate on the support of electrophoresis tank and fix the glass plate
 +
 +
2) Equipped with 12% separating glue
 +
 +
3) After the separation glue is configured, immediately pour it into the electrophoresis tank between the two glass plates, stop about 3cm away from the upper mouth, and then add a thin layer of water for water sealing for about 40 minutes, waiting for the glue to condense naturally. After condensation, pour out distilled water to absorb the water.
 +
 +
4) Equipped with 4% condensation glue
 +
 +
5. Electrophoresis
 +
 +
1) Connect the electrophoresis tank to the power supply of the electrophoresis instrument and start electrophoresis. First, the constant voltage is 80V. After the sample enters the separation gel, the constant voltage is 120V
 +
 +
2) When bromophenol blue moves to the leading edge, cut off the power supply and stop electrophoresis
 +
 +
6. Dyeing
 +
 +
1)Preparation of dyeing solution:
 +
 +
0.03g Coomassie Brilliant Blue + 7.5ml isopropanol + 3ml acetic acid
 +
 +
2)dyeing
 +
 +
Remove the gel from the electrophoresis tank, carefully remove the gel, immerse the gel in dyeing solution for half an hour.
 +
 +
7. Decolorization
 +
 +
1) Preparation of decolorizing solution:
 +
 +
15ml isopropanol + 6ml glacial acetic acid + 39ml water
 +
 +
2) Decolorization: decolorize until the strip is clear
  
  

Latest revision as of 17:15, 20 October 2021

DnrF for coding

The DnrF gene, responsible for conversion of aklavinone to ɛ-rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin (Dnr) gene cluster of Streptomyces peucetius ATCC 29050. The DnrF gene was sequenced and should encode a protein of 489 amino acids with a molecular mass of 52 kDa.[1] It is used as monooxygenase in our design which can finally let dcll become carminic acid.

Biology and Usage

DnrF involved in the biosynthesis of the anthracyclines carminomycin, rhodomycin, daunorubicin (daunomycin) and doxorubicin (adriamycin) which are aromatic polyketide antibiotics that exhibit high cytotoxicity and are widely applied in the chemotherapy of a variety of cancers. Catalyzes the incorporation of a hydroxyl group at position C-11 of aklavinone, resulting in epsilon-rhodomycinone.[2]

DnrF can convert aklabinone to ɛ-rhodomycinone via C-11 hydroxylation. The heterologous expression of DnrF in Escherichia coli demonstrated that the gene encodes a NADPH-dependent hydroxylase catalysing the hydroxylation of aklavinone to yield ɛ-rhodomycinone. It is used as monooxygenase in our design which can finally let dcll become carminic acid.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 508
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 508
    Illegal NotI site found at 1219
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1074
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 508
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 508
    Illegal AgeI site found at 722
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental approach

1. Pre growth

1) Prepare 50mg / ml streptomycin mother liquor, filter and sterilize with filter membrane

2) Add 5ml LB liquid medium into 10ml centrifuge tube and 2ul streptomycin

3) Scrape some agar (including bacteria) along the puncture line with the inoculation ring, and extend the inoculation ring into the LB medium in the corresponding centrifuge tube to complete the inoculation

4) 37 ℃ 220 RPM overnight grow

2. Expression of protein

1） Add 10ml LB medium into 50ml centrifuge tube and 4ul chloramphenicol. Make two tubes for each plasmid, one of which is the control

2) 500ul of bacteria cultured overnight were added to 10ml LB medium and expanded at 37 ℃ and 220rpm for 2.5h

3） 0.1M IPTG 50ul (final concentration: 0.5mm) was added to the LB medium of the induction group of two plasmids, which was induced at 37 ℃ and 220rpm for 4H; The control group of the two plasmids did not add IPTG, and the parallel experiment was carried out

4） Suck 1ml bacterial solution from the four cultures, add it to 1.5ml EP tube, centrifuge at 12000rpm for 1min, discard the supernatant, add 50ul ddH2O to resuspend the bacteria, and then add 50ul SDS loading buffer

5） The mixed bacteria and loading buffer were heated at 100 ℃ for 10 min

6） Put the heated protein into - 20 ℃ for later use.

3. protein extraction

1) Draw 1ml bacterial solution from the cultures of the experimental group and the control group of each strain, add it to the 1.5ml EP tube, centrifuge at 12000rpm for 1min, discard the supernatant, add 50ul ddH2O to resuspend the bacteria, and then add 50ul SDS loading buffer

2) The mixed bacteria and loading buffer were heated at 100 ℃ for 10 min

3) Put the heated protein into - 20 ℃ for standby

4. SDS polyacrylamide gel Preparation for protein detection

1) Put the dried glass plate on the support of electrophoresis tank and fix the glass plate

2) Equipped with 12% separating glue

3) After the separation glue is configured, immediately pour it into the electrophoresis tank between the two glass plates, stop about 3cm away from the upper mouth, and then add a thin layer of water for water sealing for about 40 minutes, waiting for the glue to condense naturally. After condensation, pour out distilled water to absorb the water.

4) Equipped with 4% condensation glue

5. Electrophoresis

1) Connect the electrophoresis tank to the power supply of the electrophoresis instrument and start electrophoresis. First, the constant voltage is 80V. After the sample enters the separation gel, the constant voltage is 120V

2) When bromophenol blue moves to the leading edge, cut off the power supply and stop electrophoresis

6. Dyeing

1)Preparation of dyeing solution:

0.03g Coomassie Brilliant Blue + 7.5ml isopropanol + 3ml acetic acid

2)dyeing

Remove the gel from the electrophoresis tank, carefully remove the gel, immerse the gel in dyeing solution for half an hour.

7. Decolorization

1) Preparation of decolorizing solution:

15ml isopropanol + 6ml glacial acetic acid + 39ml water

2) Decolorization: decolorize until the strip is clear


References

[1] Filippini, S & Solinas, M & Breme, U & Schlüter, M & Gabellini, D & Biamonti, Giuseppe & Colombo, A & Garofano, Luciano. (1995). Streptomyces peucetius daunorubicin biosynthesis gene, dnrF: Sequence and heterologous expression. Microbiology (Reading, England). 141 ( Pt 4). 1007-16. 10.1099/13500872-141-4-1007. [2] Hong, Young-Soo & Hwang, Cheol & Hong, Soon-Kwang & Kim, Young Ho & Lee, Jung Joon. (1994). Molecular cloning and characterization of the aklavinone 11-hydroxylase gene of Streptomyces peucetius subsp. caesius ATCC 27952. Journal of bacteriology. 176. 7096-101. 10.1128/jb.176.22.7096-7101.1994.