Applications

One Example -- the regulation of Fatty Acid synthesis

As we have emphasized at the very beginning, it is our final goal to achieve in vivo metabolic flux regulation with our gear box. To get closer to this goal, we changed the sgRNA sequence downstream of the bluelight-regulating promoter pFixK2 and targeted it at FadD, one of the genomic genes in E.coli. In this way, we had constructed a FadD regulating system, which is controlled by blue light signals, to qualitatively testify that our system can be immediately put into applications——to regulate metabolic fluxes in vivo.

About FadD

FadD is the acyl coenzyme A (CoA) synthetase in E. coli responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs (Steen et al., 2010). According to some studies,E.coli strains with a FadD mutation will accumulate more fatty acid than the normal strains, and further researches has pointed out that this enzyme is required for the upregulation of genes involved in fatty acid degradation (Yu et al., 2011). For this reason, we simply changed the base-pairing region of the sgRNA sequence in the bluelight-controlled CRISPRi system to targeted it at the FadD gene that resides on the genome of E.coli——that is to say, we didn't overexpress any extra FadD enzymes through plasmid transformation or chemical induction. So will we get an obvious change in fatty acid production if we interrogate the expression of FadD gene in vivo?

Methods

To testify our system, we set 3 groups of e.coli and culture them under the same conditions except the light intensity. one group is in total dark surrounding, one receives weak blue light and another is exposed to the full light. The blue light controlled CRISPRi system is used in this test with a self-designed sgRNA targeting towards FadD. We predict that if there is blue light, due to the decrease of the sgRNA amount, more FadD will function in cell and the fatty acid synthesis will decrease as a result.

Results

After a series of data collection work, we get the following results that are in accordance with our expectation. And the total fatty acid amounts are shown below.

From this result we can draw the conclusion that our system performs very well in this test. Due to the complicated culturing conditions and the extracting process of fatty acid both from the culture and cell itself, limited time is left for us to find out more data to further test our system. In the future we plan to repeat this test and try to perform this experiment under more different light intensities so as to get more convincing data to show the huge potential of new application of our project.

References

STEEN, E. J., KANG, Y., BOKINSKY, G., HU, Z., SCHIRMER, A., MCCLURE, A., DEL CARDAYRE, S. B. & KEASLING, J. D. 2010. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature, 463, 559-562.

YU, X., LIU, T., ZHU, F. & KHOSLA, C. 2011. In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 108, 18643-18648.