Difference between revisions of "Part:BBa K1062000"
| (6 intermediate revisions by one other user not shown) | |||
| Line 2: | Line 2: | ||
<partinfo>BBa_K1062000 short</partinfo> | <partinfo>BBa_K1062000 short</partinfo> | ||
| − | This part is a guide RNA (gRNA) that targets RFP, when bound to Cas9. It also contains a cleavage site recognized by the Csy4 enzyme which allows it to be cleaved from neighboring DNA/RNA. | + | This part is a guide RNA (gRNA) that targets the RFP gene, when bound to Cas9. It also contains a cleavage site recognized by the Csy4 enzyme which allows it to be cleaved from neighboring DNA/RNA. |
| Line 12: | Line 12: | ||
==='''Usage and Biology'''=== | ==='''Usage and Biology'''=== | ||
In both of our projects, we used gRNAs and dCas9 as: | In both of our projects, we used gRNAs and dCas9 as: | ||
| + | |||
1) To specifically target and repress the "virulence gene" (we used flourescent proteins as a proof of concept) in our conjugation project | 1) To specifically target and repress the "virulence gene" (we used flourescent proteins as a proof of concept) in our conjugation project | ||
| + | |||
2) Improve past circuits with better decision making by using dCas9 instead of traditional repressors. | 2) Improve past circuits with better decision making by using dCas9 instead of traditional repressors. | ||
| + | |||
| + | |||
| + | ==='''Characterization'''=== | ||
| + | |||
| + | We validated that this part works by testing it in our designed CRISPRi system. The RFP guideRNA (RFPi or RFP-gRNA) was transformed into a strain of JM109 containing RFP in its genome. The RFPi was on a plasmid also containing dCAS9 the protein necessary for targeting to and repression of the RFP. | ||
| + | <html> | ||
| + | |||
| + | <img align="center" style="margin-bottom:20px; width: 500px; margin-top:20px; padding:2; margin-left:55px;" src="https://static.igem.org/mediawiki/igem.org/b/bc/Direct_Transformation.jpg"> <br> | ||
| + | </html> | ||
| + | |||
| + | The efficiency of CRISPRi system measured by directly transform conjugative plasmid that contains CRISPRi system (sgRNA and dCas9 protein) for RFP into JM109 strain that have RFP inserted into its chromosome. The dCas9 protein of the repression system was under control of a pTET promoter and the sgRNA of the system was under control of a constitutively expressed promoter. Positive control was directly transform of conjugative plasmid backbone (pARO190 without sgRNA and dCas9) into JM109 strain that have RFP inserted into its chromosome, and negative control was directly transform of conjugative plasmid that contains CRISPR/Cas repression system (sgRNA and dCas9 protein) for RFP into JM109 strain without any fluorescent markers. RFP level were measured by flow cytometry and error bars showed standard deviation calculated on the basis of technical replicates. | ||
| + | |||
| + | |||
| + | =='''Conjugation Project'''== | ||
| + | |||
| + | [[File:Conjugation project.jpg]] | ||
| + | |||
| + | In the Conjugation Project, our target contained a RFP cassette that is knocked into the chromosome of the strain. Due to the fact that the target strain is essential to proving the Conjugation Project, this gRNA is essential in order to prove that repression of a specific target can be possible. | ||
| + | |||
| + | =='''Synthetic Circuit'''== | ||
| + | |||
| + | [[File:Synthetic Circuit.jpg]] | ||
| + | |||
| + | In the Synthetic Circuit, the target is the RFP gene that would be located in the '''High Concentration Pathway'''. The purpose of the gRNA is to prevent any possibility of the RFP gene expressing, due to a possible leaky promoter, when the '''Low Concentration Pathway''' is being induced. | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
Latest revision as of 19:37, 27 September 2013
Guide RNA (gRNA) target for RFP
This part is a guide RNA (gRNA) that targets the RFP gene, when bound to Cas9. It also contains a cleavage site recognized by the Csy4 enzyme which allows it to be cleaved from neighboring DNA/RNA.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
In both of our projects, we used gRNAs and dCas9 as:
1) To specifically target and repress the "virulence gene" (we used flourescent proteins as a proof of concept) in our conjugation project
2) Improve past circuits with better decision making by using dCas9 instead of traditional repressors.
Characterization
We validated that this part works by testing it in our designed CRISPRi system. The RFP guideRNA (RFPi or RFP-gRNA) was transformed into a strain of JM109 containing RFP in its genome. The RFPi was on a plasmid also containing dCAS9 the protein necessary for targeting to and repression of the RFP.
The efficiency of CRISPRi system measured by directly transform conjugative plasmid that contains CRISPRi system (sgRNA and dCas9 protein) for RFP into JM109 strain that have RFP inserted into its chromosome. The dCas9 protein of the repression system was under control of a pTET promoter and the sgRNA of the system was under control of a constitutively expressed promoter. Positive control was directly transform of conjugative plasmid backbone (pARO190 without sgRNA and dCas9) into JM109 strain that have RFP inserted into its chromosome, and negative control was directly transform of conjugative plasmid that contains CRISPR/Cas repression system (sgRNA and dCas9 protein) for RFP into JM109 strain without any fluorescent markers. RFP level were measured by flow cytometry and error bars showed standard deviation calculated on the basis of technical replicates.
Conjugation Project
In the Conjugation Project, our target contained a RFP cassette that is knocked into the chromosome of the strain. Due to the fact that the target strain is essential to proving the Conjugation Project, this gRNA is essential in order to prove that repression of a specific target can be possible.
Synthetic Circuit
In the Synthetic Circuit, the target is the RFP gene that would be located in the High Concentration Pathway. The purpose of the gRNA is to prevent any possibility of the RFP gene expressing, due to a possible leaky promoter, when the Low Concentration Pathway is being induced.