Team:SHSBNU China/Description

• Acknowledgement
• Team
  Team Members Our School Collaborations Attributions
• Judging
  Judging Achievements
• Model
  Modeling Software
• Safety
• Human Practices
  Intergrated Human Practices Public Engagement
• Parts
  Overview Basic parts Composite Part Collection Improve
• Lab
  Experiments Notebook Protocols
• Project
  Inspiration Description Design Results Hardware Demonstrate Prospect

Project Inspiration & Description

Inspiration

Inflammatory Bowel Disease (IBD) is an umbrella term used to describe the chronic inflation of digestive tract. IBD includes two types — Ulcerative colitis (UC) and Crohn’s disease (CD) (Mayo Clinic, 2017). Both US and CD can cause certain disorders, such as eye or skin inflammation, and could increase the risk of colon cancer. For the difference, UC may cause toxic megacolon and eventually result in a colon perforation. CD can affect the thickness of the colon wall, specifically narrow and thicken the bowel which may block the digestive contents (Daniel K, Podolsky M.D., 1991). Mesalazine is an efficient treatment for UC, while antibiotics have a better effect on Crohn’s disease. Anyhow, these ways inevitably have some side effects to human.

This year, our project design was inspired by the project of our school's iGEM team in 2017. They designed a probiotic which contained ThsS/R plasma. If the patient ate the probiotics, they can tell if they have IBD by observing the color of their feces. However, the project has some limitations, the results of which can only be qualitatively analyzed but not quantitatively analyzed. To be more specific, the patients can only know whether they are sick or not, but there is no way for them to know how serious their illness is. Therefore, in order to make this project more applicable, our iGEM team in 2019 will work to detect intestinal inflammation more accurately and show the results in different degrees, so as to achieve a perfect combination of quantitative and qualitative. This project can give patients with intestinal inflammation a more accurate and reliable data, so that patients and doctors can better understand the severity of their disease. Therefore, we design the bacteria that can imitate signal and record the memory, the ways to read out the memories and transfer it to quantitative signals and hardware such as capsule, which makes our recollect operation more convenient.

Description

In order to show the results seriousness of intestinal imflammation, we first chose SCRIBE. The wild-type (WT) retron cassette encodes three components in a single transcript: a reverse transcriptasesa (RTs) and two RNA moieties, msr and msd, which act as the primer and the template for the reverse transcriptase, respectively. The msr-msd sequence in the retron cassette is flanked by two inverted repeats. Once transcribed, the msr-msd RNA folds into a secondary structure guided by the base pairing of the inverted repeats and the msr-msd sequence. The RT recognizes this secondary structure and uses a conserved guanosine residue in the msr as a priming site to reverse transcribe the msd sequence and produce a hybrid RNA-ssDNA molecule called msDNA. Two stop codons were then introduced into the genomic GFP to make incapable of luminescence GFPOFF reporter strain. These premature stop codons could be reverted back to the normal GFP sequence via recombination with engineered ssDNA (GFP)ON, thus re-luminescence. The intracellularly expressed ssDNAs can be recombined into target genomic loci by concomitant expression of Beta. However, in the end, we found that the efficiency of SCRIBE transformation was very low. Thus, on the one hand, we try to improve scribe continuously, and strive to improve its efficiency. On the other side, we are also looking for new technology, CAMERA.

CAMERA

After that, we found a very efficient system. That is CAMERA. When receiving the thiosulfate signal from the intestine, the cells express Cas9 protein and single guide RNA (sgRNA) to change the sequence of the report gene. Once sgRNA successfully binds to the corresponding DNA target site, the Cas9 can edit the target sequence. Cas9 protein fuses a cytidine deaminase, which can introduce single C·G-T·A mutations without causing DNA breakage and then form a terminator, vice versa. The target sequence is 20bp close to NGG, and one of the five, six and seven bases of the target sequence must be cytidine, because cytidine deaminase has specificity.

References

Daniel, K and Podolsky, M.D. 1991. Inflammatory Bowel Disease (Second of Two Parts). The New England Journal of Medicine. 325(14), pp. 1008-1016.

Mayo clinic staff. 2017. Mayo Clinic.[Online]. [19 October 2019]. Available from: https://www.mayoclinic.org/diseases-conditions/inflammatory-bowel-disease/symptoms-causes/syc-20353315#targetText=Inflammatory%20bowel%20disease%20(IBD)%20is,intestine%20(colon)%20and%20rectum.

Fahim, F and Timothy, K. 2014. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations. Science. 346(6211), pp. 825-834.

Weixin, T and David, B. 2018. Rewritable multi-event analog recording in bacterial and mammalian cells. Science. 360(6385), pp. 1-10.