Team:Peking/Perspective
Future Plan
Confirm the effects of CRISPR/dCas9 system on different cells by RNA-seq
In our experiment, inhibition of bacterial genomic DNA replication is achieved by dCas9 binding to the Ori region. But we are not aware of the physiological state of the bacteria. Therefore, we will verify the physiological state of the bacteria by means of RNA-seq.
Many studies indicate that in many Gram negative bacteria, several envelope stress response, including two-component regulatory system (TCRS), of E. coli are involved in the maintenance, adaption and protection of the bacterial cell wall in response to variety of stress. Another aspect of stress response is the generation of morphological modifications. Generally speaking, bacterial morphology will change as growth conditions change.Any modification in cell shape is connected with cell wall metabolism and require specific regulatory mechanisms. Recent advances support the existence of complex mechanisms mediating morphological responses to stress involving inter and intra-specific signaling. [1]
We found that the bacteria transferred to CRISPR/dCas9 system become longer than the normal bacteria. So we will focus on the mRNA about two-component regulatory systems (TCRS) and their activating signals.
Transfer the drug synthesis pathway into engineering strains to verify the yield increase effect
Our project aims to build an optimized medical engineering bacteria, so we will transfer the drug synthesis pathways such as low toxicity protein and some interferon gene [2] into the engineered bacteria and verify the activity and yield of the synthetic drug.
Experiment with different strains to verify the versatility of the system
In our experiments, we only verified the feasibility of our CRISPRri system in E. coli. We will transfer our CRISPRri system to different strains to verify the versatility of this system. If we can produce similar effects in a large number of strains, then a more general DNA replication regulatory system can be realized.
In addition, the indigo characterization experiment and model validation can indicate an increase in the yield of the target product in the bacteria transferred to our CRISPRri system.
Biosynthetic production is widely used in life. In terms of food production, bacteria can produce a variety of enzymes, amino acids, flavorings and other compounds used in food production; in industrial production, bacteria can produce many recombinant proteins, dyes, biofuel[3], etc; in pharmaceutical production, it is now available for production of insulin proteins[4] to treat diabetes, clotting factors to treat haemophilia[5], human growth hormone, erythropoietin for anemic patients and interferon for the treatment of cancer by bacteria [6]. By introducing our CRISPRri system to bacteria that synthesize different product , we can achieve an increase in the yield of the target product and thereby applying for more scenes.
Combined with phase separation to verify whether the proliferation rate can be increased by recruiting DnaA protein to near Ori.
A complete set of DNA replication regulatory systems should include inhibitory and enhanced effects, so we will combine phase separation related techniques to increase DNA replication. In the 2018 Peking University iGEM project, they designed a modular and flexible system. An intracellular stable phase is formed by contacting proteins and proteins with each other.(see Peking IGEM 2018)Controllable protein phase separation and modular recruitment can form responsive membrane-less organelles and result in colocalizing molecules at high concentrations.[7] In this way, we can promote the replication of DNA by accumulating DnaA protein adjacent to Ori to promote the binding of DnaA to DNA
Bacteria transferred into CRISPR/dCas9 system can reduce horizontal gene transfer
We observed that some bacteria produced by the division of longer bacteria can continue to divide, while a small number of bacteria will no longer divide or even grow. So we assume that part of the split bacteria does not contain the bacterial genome, thus preventing horizontal gene transfer [8]. So it can play an important role in biomedical and scientific research.
Reference：
1、 Jose M. Requena(2012), Stress Response in Microbiology,
2、 Henri Braat(2006), A Phase I Trial With Transgenic Bacteria Expressing Interleukin-10 in Crohn’s Disease, CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2006;4:754–759
3、 Summers, Rebecca (24 April 2013) "Bacteria churn out first ever petrol-like biofuel" New Scientist, Retrieved 27 April 2013
4、 Walsh G (April 2005). "Therapeutic insulins and their large-scale manufacture". Applied Microbiology and Biotechnology. 67 (2): 151–9. doi:10.1007/s00253-004-1809-x. PMID 15580495.
5、 Pipe SW(May 2008). "Recombinant clotting factors". Thrombosis and Haemostasis. 99 (5): 840–50. doi:10.1160/TH07-10-0593. PMID 18449413.
6、 Jumba M (2009). Genetically Modified Organisms the Mystery Unraveled. Durham: Eloquent Books. pp. 51–54. ISBN 9781609110819.
7、 Benjamin S. Schuster(2018), Controllable protein phase separation and modular recruitment to form responsive membraneless organelles, NATURE COMMUNICATIONS, DOI: 10.1038/s41467-018-05403-1
8、 Reardon S (June 2018). "Genetically modified bacteria enlisted in fight against disease". Nature. 558 (7711): 497–498. doi:10.1038/d41586-018-05476-4. PMID 29946090.