Part:BBa_K4662049
sgRNA to block Beta-galactosidase
The sgRNA construtct can be used to block beta-galactosidase when dCas9 is expressed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
LacZ sg RNAs were tested by co transformation with pFD152 a plasmid containing dCas9 under the expression of a Tetracycline dependent promoter.
Blue white screen was repeated several times using different batches of X-Gal plates and different levels of tetracycline as well as different methods for induction by tetracycline. However, colonies always appeared blue.
Figure 1: Unsuccessful induction of the LacZ sgRNA to block the beta-galactosidase.
Conclusion
Blue white screen of LacZ sgRNAs could have failed due insufficient activation of the Tet promoter that regulates dCas9. To ensure sufficient activation, anhydrotetracycline which shows a stronger binding affinity and is not toxic to bacteria could be used to induce dCas9 expression. However it is also likely that the LacZ sgRNAs performed poorly.
Usage and Biology
sgRNA construct can be used for Blue-white screening
Remark
Specific protocols for assembly and testing can be found on our wiki https://2023.igem.wiki/uzurich/experiments
If any questions arise or additional information is needed, we can provide a more extensive list of experiments that we can share if required.
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