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.

New Part: BBa_K1689013

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.

We used Microcystin-LR detection kit (WRZ001) from 天河綠洲. We believed that our dCas9-sgRNA complex has been successfully been expressed. This is because the Microcystin-LR concentration in our transformed Microcystis was lower than 0.002mg/L, while our control set ups have a higher concentration.

Fig 1. Microcystin detection kit sample

Fig2. 1st test (from left): Culture of Microcystis 3 weeks after transformation
2nd test: Water
3rd test: Culture of unsuccessful Microcystis transformation after 3 weeks/br> 4th test: Positive control of Microcystis culture that has not been transformed

The successful transformation of Microcystis culture showed Microcystin concentration less than 0.002mg/L. This shows a decrease in Microcystin toxin.

References

GFP Expression
Through UV excition, we the green flourescence protein has been successfully expressed, demonstrating flourescence properties.
Fig3. Cell cultures with disired plasmid under UV excitation.

[1]

Larson, M. H. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, 2180–2196.

Team:HK SSC/Improve

Improve a Previous Part or Previous Project

Original Part BBa_K1689013

New Part: BBa_K1689013

Results

References

GFP Expression