Current Results

Double Promoter
We have successfully ligated tandem double promoters with J23102 J23106 and J23116 in different orders: J23102-J23102, J23102-J23106, J23106-J23102, J23116-J23102, J23106-J23106, J23106-J23116, J23116-J23106. And we have submitted these new biobrick. Unfortunately, other combination failed because of mutation showed by sequence result.
Then we transfer the double tandem promoters with the reporter gene RFP to the expression vector pSB2K3. Through the analysis of the fluorescence/OD600 ratios,we identify the relevant transcription strength of each tandem promoter.
We measured the specifc strength of double promoters according to the fluorescence of reporter gene. Our results show that different combinations of double promoters have disparate transcriptional strength , in which the J23102-J23102 combination ranks as the best one. More importantly, once combined together, double promoters have stronger transcriptional strength compared to that of respective single promoter. In our essay, B0034 was used as a standard RBS module.


Guide RNA
We chose tandem double promoters J23106-J23116 as our target. Based on this, we designed serval targeting sites, including both activation(A1,A2,A3,A4,A5) and repression(R1, R2) sites after searching the PAM region. And through a three-cycle overlap PCR, we construct these functinal guide RNA.

Here shows the agarose electrophoresis:


dCas9 Devices
First, we add dCas9 gene( amplify through PCR) to express vector with promoter to construct a new biobrick. We name this new biobrick BBa_K1081000.


Here shows the agarose electrophoresis and SDS-PAGE of BBa_1081000:


Multi-stage regulation results
After construction of all the three components,tandem promoter, guide-RNA and dCas9, we can achieve our final goal—— multi-stage regulation of the target gene expression. Here we show some results of our experiments.

In the following figure, BBa_J23106 represents promoter 1 and BBa_J23116 represents promoter 2.

In the following figure, BBa_J23106 represents promoter 1 and BBa_J23102 represents promoter 2.
aCas9 Devices
Then we ligate omega subunit gene downstream of dCas9.
Here shows the agarose electrophoresis of the product:

Future Work

After long terms of hard work, we have successfully achieved some of our expected results, including tandem promoter system and preconceived dCas9 targeting system. Although we are passionate enough to accomplish what we have planned previously, constraints come from diverse aspects which impede us from doing so. Time is limited, so do our academic experiences. Under such circumstances, we still have much to do concerning with our novel multi-level gene expression regulator system in future as follows.

1. Tandem Promoter System
Even regarded as the foundation of multi-level gene expression regulation, functions of tandem promoter system will not be limited as we have described on our wiki. Our temporal project has only included double promoters. What we have in mind for future work are to increase the number and type of promoters used in our present system.



2. dCas9 Activation Platform
In our current experimental results, only parts of our preconceived dCas9 targeting system have been accomplished. However, based on previous research, we have designed several novel activation system via disparate protein-protein interactions.



First, four kinds of protein-protein interactions are designed as follows. Both dCas9 protein and alpha subunit of RNAP are fused with Gal11/CI repressor and Gal4-1/Gal4-2/VP16/CI repressor respectively. In other words, Gal11 protein interacts with Gal4-1 ,Gal4-2 and VP16 protein while dCas9-CI repressor complex can recruit RNAP through CI-CI interaction. Our goals can be eventually fulfilled when RNAP is recruited by relevant dCas9-protein complex and downstream gene expression is up-regulated as expected.

Second, based on previous research that omega subunit of RNAP can also be fused with dCas9 protein to activate gene expression, we further devise a protein-protein interaction between dCas9-CI repressor complex and CI-omega complex of which the strength of activation may be even higher compared to pure dCas9-omega and RNAP interaction system.

Probably, we may finally accomplish a much better gene activation system compared to existed/reported activation systems via such dCas9 activation platform as stated above. Futhermore, the tandem promoter system and dCas9 activation system can be combined again to achieve multi-level gene expression regulation. Thus, once given enough time, we are sure that much better achievements can be made in the long run.