Part:BBa_K3219002
Plasmid for in vivo expression of dCas9-sgRNA targeting mcyB
Microcystis Aeruginosa is a noxious cyanobacterium that produces a hepatotoxin, Microcystin. This plasmid silences the McyB gene of Microcystis Aeruginosa UTEX2388 using the dCas9 and sgRNA. According to previous researchers, Microcystin cannot be produced without McyB[1]
This plasmid can be used for in vivo or in vitro expression of dCas9 and sgRNA.
This plasmid consists of a cyanobacteria shuttle vector, ribosome binding sites, a dCas9-GFP complex and a sgRNA targeting mcyB of Microcystis Aeruginosa UTEX 2388 McyB.
Shuttle Vector
The shuttle vector is from team 2016 iGEM team Nanjing_NFLS, part BBa_K1894001. It consists of two origin of replications, f1 ori and pUC ori, which allows it to replicate in both E.coli cells and cyanobacteria cells. It also consists of a t7 promoter, CaMV35S promoter, and Kanamycin resistance gene. The T7 promoter allows expression of the dCas9-GFP-sgRNA in E.coli and the CaMVS promoter allows expression in cyanobacteria.
dCas9
The dCas9, also known as a catalytically dead Cas9 enzyme, is a mutated Cas9 enzyme without endonuclease activity. With the help of a single-guide RNA (sgRNA), it specifically binds to the target sequences and blocks transcript elongation by RNA polymerase. The GFP is added to the C-terminus of the dCas9, connected using a linker. There is also a 7x His-Tag, allowing for protein purification.
sgRNA
The sgRNA consists of a handle, a base-pairing region and a terminator. The base-pairing region is designed to target 25 base pairs of the McyB gene of Microcystis Aeruginosa UTEX2388.
Usage
This part can be used by transforming it directly into Microcystis Aeruginosa UTEX 2388 via electroporation or naturual transformation. Our team used the protocols provided by Au Yang Qin [2], Nermin Adel El Semary[3] and Elke Dittmann[4]. After the expression of dCas9 and sgRNA will result in the repression of the McyB gene. Thus, no Microcystin will be produced.
Characterization
Introduction
We would like to see how the transformation of this plasmid affects the growth of cells. We transformed our part (BBa_K3219002) into E.coli BL21 and E.coli DH5α, and compared their growth rates to E.coli transformed with common plasmids. In this case, we can see how this large plasmid of 12kbp affects the growth rate of cells, and whether it should be modified in future studies.
Background:
It was found that plasmid sizes and different origin of replications have distinct effects on the growth of E.coli [5]. Generally, larger plasmid sizes result in a slower growth rate in E.coli. While our plasmid is larger than 12 kbp, we would like to know whether this large plasmid size will affect the growth rate of E.coli transformed with this plasmid. If the transformation of this plasmid results in a very low cell yield or a slow growth curve, protocols may have to be modified in order to achieve ideal plasmid yield (for conducting plasmid purification) or protein expression.
Methods:
1. Preparation of Cells
E.coli competent cells were prepared using Inoue Method[6] .
2. Calibration
We followed iGEM 2019 Plate Reader Abs600 (OD) Calibration protocol, s that we can estimate our number cells.
3. Design and Cloning of BBa_K1894002
We designed
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3964
Illegal NheI site found at 12275
Illegal NotI site found at 777 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1685
Illegal XhoI site found at 716 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 7979
Illegal NgoMIV site found at 8139
Illegal NgoMIV site found at 11186 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 5721
Illegal SapI site found at 9059
- ↑ Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.
- ↑ 章军, 徐虹, 楼士林, 欧阳青. Blue-green alga shuttle plasmid expression vector and method for expressing thymison 'alpha' 1. Thesis. Xia Men: Xia Men University, 1999.
- ↑ Semary, Nermin Adel El. “Optimized electroporation-induced transformation in Microcystis aeruginosa PCC7806.” Biotechnol. Agron. Soc. Environ (2010): 149-152 . Journal.
- ↑ Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.
- ↑ U. EONG CHEAH, WILLIAM A. WEIGAND, BENJAMIN C. STARK. “Effects of Recombinant Plasmid Size on Cellular Processes in Escherichia coli.” Plasmid (1987): 127-134 . Journal.
- ↑ Im, H. (2011). The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells. Bio-101: e143. DOI: 10.21769/BioProtoc.143.
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