Difference between revisions of "Part:BBa K3219000"
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We have successfully silenced the McyB gene in Microcystis Aeruginosa UTEX 2388 using this construct. We cloned BBa_K3219000 into a shuttle vector with CaMV35S RNA promoter and ribosome binding site. After 3 weeks after transformation of the shuttle vector with part BBa_K3219000, the Microcystin concentration was lowered compared to the control set-ups. <br> | We have successfully silenced the McyB gene in Microcystis Aeruginosa UTEX 2388 using this construct. We cloned BBa_K3219000 into a shuttle vector with CaMV35S RNA promoter and ribosome binding site. After 3 weeks after transformation of the shuttle vector with part BBa_K3219000, the Microcystin concentration was lowered compared to the control set-ups. <br> | ||
[[File:T--HK_SSC--Test_paper.jpg|500px|thumb|center|Figure 1: Microcystin detection kit sample]]<br> | [[File:T--HK_SSC--Test_paper.jpg|500px|thumb|center|Figure 1: Microcystin detection kit sample]]<br> | ||
| − | + | [[File:T--HK_SSC--results.jpeg|500px|thumb|center|Figure 2: Our results 1st test (from left): Culture of Microcystis 3 weeks after transformation<br> | |
| + | 2nd test: Water<br> | ||
| + | 3rd test: Culture of unsuccessful Microcystis transformation after 3 weeks<br> | ||
| + | 4th test: Positive control of Microcystis culture that has not been transformed<br>]]<br> | ||
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Revision as of 14:16, 20 October 2019
dCas9-GFP
This is a part improvement of BBa_K1689013 (https://parts.igem.org/Part:BBa_K1689013).
Original Part BBa_K1689013
Part BBa_K1689013 is an N-terminal fragment of β-lactamase fused with dCas9. Team iGEM15_Peking designed it to be resistance against several antibiotics. However, β-lactamase may not be applicable to each and every project. For example, in our project, the plasmid already confers Kanamycin resistance gene. β-lactamase may not be applicable in this situation. Other than using β-lactamase as a selection method, we hope to provide more options for CRISPR imaging. The GFP will allow visual confirmation of successful transformation and indicates that the dCas9 enzyme has been successfully expressed.
BBa_K3219000
dCas9 enzyme is also known as a catalytically dead Cas9 enzyme[1]. Different from traditional CRISPR Cas9 enzymes, dCas9 lacks endonuclease activity. It does not cleave DNA. Instead, with the help of a guide RNA, it specifically binds to the target, usually 20 -30 bp, and blocks transcript elongation by RNA polymerase.
In this part, a GFP is added to the C-terminus of the dCas9, connected using an SGAAAAGGS linker. The GFP is added so that the expression of both proteins could be checked easier.
We did not add ribosome binding sites or promoters to this sequence to allow larger flexibility for users to choose the promoter and RBS that is suitable for their chassis.
Results
We have successfully silenced the McyB gene in Microcystis Aeruginosa UTEX 2388 using this construct. We cloned BBa_K3219000 into a shuttle vector with CaMV35S RNA promoter and ribosome binding site. After 3 weeks after transformation of the shuttle vector with part BBa_K3219000, the Microcystin concentration was lowered compared to the control set-ups.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1837
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4116
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 644
- ↑ Larson, M. H. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, 2180–2196.