Difference between revisions of "Part:BBa K4033008"
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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
 +
 +
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 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
 +
  
  

Revision as of 17:25, 19 October 2021


antE

AntE is extracted from Photorhabdus luminescens. It is a component of the type II PKS system that can produce octaketides in E.coli.

Biology and Usage

The entire mPKS complement from P. luminescens, comprising the KS AntD, CLF AntE, and ACP AntF, were successfully expressed as soluble recombinant proteins in E. coli. Whilst AntD is a soluble recombinant proteins in E. coli, the role of AntE as a functional heterodimer partner for AntD was unknown. Sequence features of AntE defy conventions of characterised CLFs: alignment of CLF amino acid sequences in our dataset showed AntE to fringe the clade of canonical CLFs and to also lack hallmark and gatekeeper residues that play important roles in polyketide biosynthesis.

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
    COMPATIBLE WITH RFC[25]
  • 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 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



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