Difference between revisions of "Part:BBa K4033003"

Revision as of 16:52, 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) 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

@@ Line 26: / Line 26: @@
 <b><font size="3">Experimental approach</font></b>
+) 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
+) 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
+) 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
+) 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
+) The mixed thallus and the loading buffer were heated for 10min at 100℃
+) Put the heated protein into -20℃ for later use