Part:BBa_K4376003
the composite part is used to express the bpsA gene.
Construction of the bpsA gene and tac inducible promoter to control the production of the blue-pigment indigoidine synthetase. The composite part is inserted into C.glutamicum in plasmid, resulting in the successful biosynthesis of indigodine pigment.
We amplified two bpsA fragments then transformed recombinant vector into DH5α E.coli strain and Corynebacterium glutamicum.
Nanjing-China 2023's Characterization
Expression of indigoidine in Corynebacterium glutamicum
We have successfully expressed bpsA in Corynebacterium glutamicum. As shown below, the right conical flask shows the fermentation results after introducing empty PEKEX2 into the C.glutamicum, whereas the left conical flask shows the fermentation results of indigoidine production after introducing bpsA plasmid into C.glutamicum. Obviously, the left one expresses bpsA successfully with fully blue in the fermentation broth.
We used DMSO to suspend C.glutamicum, and then sonicated the bacteria to break them apart. After centrifugation, we collected the supernatant to measure the absorption peak, and the absorption peak was about 590nm, which proved that it was indeed indigoidine.
Direct Dyeing
We stained the bacterial cellulose membranes directly with C. glutamicum cultures to test the binding capacity of microbial dye to bacterial cellulose membrane.
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
[2] Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622 Fricke, P.M., Klemm, A., Bott, M. et al. On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases. Appl Microbiol Biotechnol 105, 3423–3456 (2021).
[3] Goosens VJ, Walker KT, Aragon SM, Singh A, Senthivel VR, Dekker L, Caro-Astorga J, Buat MLA, Song W, Lee KY, Ellis T. Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria. ACS Synth Biol. 2021 Dec 17;10(12):3422-3434.
[4]Florea M, Hagemann H, Santosa G, Abbott J, Micklem CN, Spencer-Milnes X, de Arroyo Garcia L, Paschou D, Lazenbatt C, Kong D, Chughtai H, Jensen K, Freemont PS, Kitney R, Reeve B, Ellis T. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proc Natl Acad Sci U S A. 2016 Jun 14;113(24):E3431-40.
[5]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.
[6]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 DyesACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622
[7]Gilbert, C., Tang, TC., Ott, W. et al. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat. Mater. 20, 691–700 (2021). Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3893
Illegal XhoI site found at 3046
Illegal XhoI site found at 3745 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 292
Illegal NgoMIV site found at 699
Illegal AgeI site found at 1957
Illegal AgeI site found at 2010 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 235
Illegal BsaI site found at 1546
Illegal BsaI site found at 2260
Illegal BsaI.rc site found at 364
Illegal BsaI.rc site found at 3550
Illegal SapI.rc site found at 1798
None |