Part:BBa_K4605003
Using strong promoters to improve the bpsA expression
Description
BpsA stands for the Blue-pigment indigoidine synthetase gene.Itself is derived from Streptomyces lavendulae and is used in the synthesis of indigo. It can only be activated from inative apo-form to the active holo-bpsA by the addition of CoA to its PCP, catalyzed by PPTase, which synthesizes two molecules of glutamine into one molecule of indigo. Corynebacterium glutamicum is usually used to express bpsA for high indigo production.
In this experiment we will modify Komagataeibacter xylinus to express bpsA for one-step synthesis of colored fibers, and also codon optimize the bpsA coding sequence.
Experiment
Promoters
We chose to use the Anderson family promoter, first, because it is compatible with K.xylinus, and there is enough references reporting that these promoters have been tested in several of bacteria such as G. xylinus 700178, G. hansenii 53582, and K. rhaeticus iGEM.Then we selected three of the strong promoters:J23100,J23104 and J23119.We linked it to bpsA-pcpS in the hope that colorful cellulose could be expressed.
Expression of bpsA in K. xylinus
With previous basic explorations, we will use the PSB1A2 plasmid backbone and strong promoters such as J23104 and J23100.Meanwhile,We constructed bpsA-pcpS gene fragments which were independently connected to plasmids by homologous recombination.The addition of pcpS makes bpsA turn into active holo-form, allowing it to be expressed smoothly in K.xylinus.
References
[1] Mohammad Rifqi Ghiffary, Cindy Pricilia Surya Prabowo, Komal Sharma, Yuchun Yan, Sang Yup Lee, and Hyun Uk Kim.High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622 [2]Teh MY, Ooi KH, Danny Teo SX, Bin Mansoor ME, Shaun Lim WZ, Tan MH. An Expanded Synthetic Biology Toolkit for Gene Expression Control in Acetobacteraceae. ACS Synth Biol. 2019 Apr 19;8(4):708-723.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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