Part:BBa_K4033003

Ant D

Ant D is extracted from Photorhabdus luminescens, and is responsible for biosynthesis of anthraquinone pigments in the nematode symbiont P. luminescens TT01.

Biology and Usage

While identifying Ant D from the anthraquinone BGC of P. luminescens TT01, phylogenetic reconstruction of the sequence alignment associate more closely with the FabF homologoues than all other KS sequences acquired from the MiBIG database. Ant D could promote the successful expression of heterologous gene in active and soluble form in E.coli. Also, Ant D is responsible of anthraquinone pigments in the nematode symbiont P. luminescens TT01, and the biosynthesis of its anthraquinone core is proposed to be enzymatically congruent with biosynthesis of actinorhodin.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 44
  
• 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 KanR. 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] : Cummings M, Peters AD, Whitehead GFS,Menon BRK, Micklefield J, Webb SJ, et al. (2019)Assembling a plug-and-play production line for combinatorial biosynthesis of aromatic polyketides in Escherichia coli. PLoS Biol 17(7): e3000347. https://doi.org/10.1371/journal.pbio.3000347