Team:SJTU-BioX-Shanghai/Parts
Parts
Overview
This year we have submitted 27 parts, with 16 basic parts and 11 composite parts. Don't think we win at quantity, we indeed provides a complete idea for designing CRISPRa and CRISPRi systems to evaluate and select dCas9 mutants that have higher on-target level and lower off-target level with a series of data with high-quality. This page provides an overview of all BioBricks that we have created and submitted to the iGEM Registry of Standard Biological Parts. For more detailed information and characterization of these parts, please use the links below to reach the Registry page of each BioBrick.
Basic part
| Partnumber | Shortdescription | Type |
|---|---|---|
| BBa_K3365000 | dCas9 protein fused with submit Omega | Coding |
| BBa_K3365001 | Target binding site for dCas9 and submit Omega | Other |
| BBa_K3365002 | RBS | RBS |
| BBa_K3365003 | Promoter of the L-arabinose operon of E. coli (pBAD) | Promoter |
| BBa_K3365006 | Target sequence downstream of pBAD/araC | Signaling |
| BBa_K3365007 | Lure1 sequence downstream of pBAD | Signaling |
| BBa_K3365008 | Lure2 sequence downstream of pBAD | Signaling |
| BBa_K3365009 | Lure3 sequence downstream of pBAD | Signaling |
| BBa_K3365011 | sgRNA production module | Other |
| BBa_K3365013 | Inducible pBAD/araC promoter | Regulatory |
| BBa_K3365052 | RFP | Coding |
| BBa_K3365055 | Lure1 sequence upstream of J23117 | Other |
| BBa_K3365056 | Lure2 sequence upstream of J23117 | Other |
| BBa_K3365057 | Lure3 sequence upstream of J23117 | Other |
| BBa_K3365058 | Lure4 sequence upstream of J23117 | Other |
| BBa_K3365059 | Kanamycin resistant gene with homologous arms | Other |
Composite part
| Part number | Short description | Type |
|---|---|---|
| BBa_K3365014 | pBAD/araC upstream of RFP with inhibition unit containing target | Composite |
| BBa_K3365015 | pBAD upstream of GFP with inhibition unit containing Lure1 | Composite |
| BBa_K3365016 | pBAD upstream of GFP with inhibition unit containing Lure2 | Composite |
| BBa_K3365017 | pBAD upstream of GFP with inhibition unit containing Lure3 | Composite |
| BBa_K3365053 | RFP with double terminator | Composite |
| BBa_K3365054 | Enhanced GFP with double terminator | Composite |
| BBa_K3365061 | Transcription activating unit upstream of RFP containing target sequence | Composite |
| BBa_K3365062 | Lure1 sequence upstream of eGFP | Composite |
| BBa_K3365063 | Lure2 sequence upstream of eGFP | Composite |
| BBa_K3365064 | Lure3 sequence upstream of eGFP | Composite |
| BBa_K3365065 | Lure4 sequence upstream of eGFP | Composite |
Part improvement
We add target sequence downstream of BBa_K1321333 created by Group iGEM14_Imperial and creat the new part BBa_K3365006
CRISPR-Cas is a prokaryotic immune system in bacteria and the function of Cas9 is to identify and bind to target site under the guidance of gRNA and make a double-stranded DNA break at target site. Inactivating both nuclease domains of Cas9 generates a catalytically dead Cas9 (dCas9) which can only bind to target site. For showing the on-target or off-target of dCas9 to further selection, we design to put the target site in the transcription unit of reporter.
For optimizing dCas9 targeting to the certain sequence, we improve the previous part BBa_K1321333 by adding a target site which can be changed to any sequence you want. We choose this inducible promoter as a part of transcription for the convenience of detecting, which means that the reporter expresses only in the presence of arabinose so you can choose a proper time to start detecting effect of transcription inhibition.
Then we use this part to construct a composite part BBa_K3365014 and insert it into a plasmid to be characterized, the result is as follow.
Part characterization
We characterized part BBa_K3365014 created by us.
As shown in the figure below, we set target sequence of dCas9 between part BBa_I0500 and reporter part BBa_K3365053. The pBAD is regulated by the AraC protein, which is both a positive and a negative regulator. The binding of dCas9 to any position within the region between the promotor and RBS might prevent transcription. Therefore, the uninduced transcriptional level of RFP is very low. In the presence of arabinose, transcription from the pBAD promoter is turned on and there will be a relatively strong fluorescence expression. In the presence of both arabinose and the complex of dCas9 and sgRNA, the complex might bind to the target sequence and the transcription is partially inhibited because of the block of RNAP. So, a relatively weak fluorescence expression of bacteria indicates a dCas9 with higher on-target rate that effectively inhibits the expression of reporter gene.
In order to characterize this part, it is transformed E. coli K12 MG1655 with BBa_K3365014 (pIn-RTr) and BBa_K3365000 (pdCas9-$\omega$). As a control, we employed E. coli K12 MG1655 carrying only one genetic circuits: BBa_K3365014(pIn-RTr) at the same time.
We took isolated colonies to pre-inoculate in 5mL of Luria-Bertani (LB) , incubating overnight in a shaker at 37℃. After that we transfer 0.5 mL of it into another 5mL of LB and then measured the Optical Density (OD) on spectrophotometer 600 nm aiming to adding arabinose between 0.4 OD and 0.6 OD. We detected the fluorescence intensity of the bacterial solution by microplate reader every hour after adding arabinose.
The result is shown as following. The binding of dCas9 can efficiently suppress the expression of RFP, which can be changed to any reporter you want.
To know about the inhibition of the expression of RFP by dCas9 in a single cell as well as verify our mutant library, the obtained dCas9 mutant library and the wild-type dCas9 are transformed into MG1655 carrying pIN-RTr. Then, we analyze the red fluorescence by flow cytometry.
The results are shown below. 77.12% cells have positive red fluorescence in the absence of dCas9, while the proportion of positive cells decreases to 0.08% in the presence of dCas9, showing a quite efficient inhibition of wild-type dCas9. And the positive rate rises to 78.37% when the cells are transformed into dCas9 mutant library, which means the lack of function in most of mutant dCas9 and the successful construction of our mutant library.
SJTU-BioX-Shanghai
contact us
Contact us: sjtuigem@gmail.com or sjtuigem@gmail.com
Bio-X Institute, Shanghai Jiao Tong University, Dongchuan Rd. 800
Copyrights © 2020 SJTU-BioX-Shanghai
