Revision as of 11:11, 17 October 2018

AraC-Pbad-lacI-sgRNA We use the lacI repression system to regular the transcription of sgRNA which is designed to specifically bind to the gene panD on the E. coli genome

Usage and Biology

We combined lacI repression system (BBa_K2556031) which is controlled by the AraC-Ara promoter (BBa_K808000) with sgRNA (BBa_K2556041) so that we can regular the transcription of sgRNA. It’s important to activate the CRISPR/Cas9 system only under certain proper conditions. When activating the AraC-Ara promoter by adding L (+)-arabinose, the lacI repressor will expressed and the transcription of sgRNA will be inhibited. But this inhibition can be relieved after induction with lactose or isopropyl-beta-D-thiogalactoside (IPTG). So when the inhibition is relieved, the sgRNA will transcribe successfully and combine with Cas9 to cut the target gene panD.

Characterize

We constructed two compatible plasmids containing the sgRNA-regulated transcriptional system (BBa_K2556042) and the cas9 protein, respectively. And then we introduced two plasmids into E.coli MG1655. After obtaining the transformants, we try to demonstrate the difference of the transcriptional level of sgRNA by analyzing the growth curve in four different conditions (①+Ara+IPTG ②+Ara ③+IPTG ④None) .

Experimental Results

Analysis of the experimental results revealed that the LacI repression system couldn’t regulate the transcription of sgRNA successfully. Because four growth curves in different conditions were exactly alike. We suspected the lac operator which directly links to the sgRNA affects its conformation so that the sgRNA can’t be unable to function properly.
After consulting the literatures, we demonstrated it was exactly a DNA sequence before the sgRNA that prevented it from maturation. And it also doesn’t work if we add a repeat artificially before sgRNA. Because it has been published that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein. This limits the use of the CRISPR/Cas9 system in E.coli. Here we presented two solutions. First, try to find a suitable repression system to replace the lacI repression system, which means sgRNA should be linked directly to the transcription initiation site (TSS). Second, activate the type I CRISPR/Cas system in E.coli because of the less limitation in comparison with the type II CRISPR/Cas system.

Sequence and Features

References

[1]Deltcheva E, Chylinski K, Sharma C M, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III[J]. Nature, 2011, 471(7340):602-607.
[2]Citorik R J, Mimee M, Lu T K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases[J]. Nature Biotechnology, 2014, 32(11):1141-1145.

Functional Parameters