Difference between revisions of "Part:BBa K4033003"
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<b><font size="3">Experimental approach</font></b>
 
<b><font size="3">Experimental approach</font></b>
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1. Pre growth
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1) Prepare 50mg / ml streptomycin mother liquor, filter and sterilize with filter membrane
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2) Add 5ml LB liquid medium into 10ml centrifuge tube and 2ul streptomycin
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 +
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
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4) 37 ℃ 220 RPM overnight grow
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 +
2. Expression of protein
 +
 +
1. Pre-growth
 +
1） Add 10ml LB medium into 50ml centrifuge tube and 4ul streptomycin. 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
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5） The mixed bacteria and loading buffer were heated at 100 ℃ for 10 min
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6） Put the heated protein into - 20 ℃ for standby
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 +
Expression of protein
  
 
1) Add 10mL of LB medium and 4ul streptomycin into a 50ml centrifuge tube. Each plasmid was put in two tubes, one of which was a control tube
 
1) Add 10mL of LB medium and 4ul streptomycin into a 50ml centrifuge tube. Each plasmid was put in two tubes, one of which was a control tube

Revision as of 17:25, 19 October 2021


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. Pre-growth 1） Add 10ml LB medium into 50ml centrifuge tube and 4ul streptomycin. 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 standby

Expression of protein

1) Add 10mL of LB medium and 4ul streptomycin into a 50ml centrifuge tube. Each plasmid was put in two tubes, one of which was a control tube 2) Add 500ul of overnight cultivated bacteria to the 10ml LB medium for extended cultivation. Put the tubes into shaker and shake at 220rpm at 37℃ for 2.5h 3) Add 50ul of 0.1M IPTG (final concentration: 0.5mm) into LB medium. Put the tubes into shaker, shake at 220rpm at 37℃ for 4h; The control group of the two plasmids was conducted in parallel without IPTG 4) 1ml of bacterial solution was absorbed from the four tubess and added into a 1.5ml EP tube, centrifuged at 12000rpm for 1min, discarded the clear solution on the top, and added 50ul ddH2O for resuspension, followed by 50ul SDS loading buffer 5) The mixed thallus and the loading buffer were heated for 10min at 100℃ 6) Put the heated protein into -20℃ for later use


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