Part:BBa_K3219000

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. 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

Gene silencing
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.

Figure 1: Microcystin detection kit sample

Figure 2: Our results:
1st test (from left): Culture of Microcystis 3 weeks after transformation
2nd test: Water
3rd test: Culture of unsuccessful Microcystis transformation after 3 weeks
4th test: Positive control of Microcystis culture that has not been transformed

These results indicate that our successful transformation of Microcytsis has a Microcystin concentration lower than 0.002mg/L, while our positive control set-ups have a higher concentration, showing positive results. The experimental set-up and control set-ups were treated under the same conditions to ensure a fair test. The results show that the dCas9 was expressed and was able to silence the McyB gene.

GFP expression
We exited the cell culture with the desired plasmid under UV light. The cell cultures demonstrated fluorescence properties.

Figure 2: Cell cultures with desired plasmid under UV light

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1837
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4116
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 644