Team:SHSBNU China/Design

• Acknowledgement
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• Judging
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• Safety
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• Parts
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• Lab
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• Project
  Inspiration Description Design Results Hardware Demonstrate Prospect

Design

Our design aims at testing chemicals by bacteria in the intestinal environment and readout the final results outside in lab. The main process is to let patient eat the capsule which contains bacteria that can record analog signals in DNA. Then, the patient can exclusively use magnet to find the capsule in his or her feces and send it back to hospital or clinic. The doctor can analyze and quantify the expression intensity of GFP to determine the severity of inflammatory bowel disease（IBD）.

In order to achieve the goal, the design should include — 1) the bacteria that can record analog signal and memorize it into DNA. 2) ways to read out the memories and transfer it to quantitative signals. 3) hardware such as capsule.

First, in terms of testing the inflammatory bowel disease molecular, we referenced 2017 SHSBNU_China team’s idea. In 2017, our school's iGEM team's task was to detect four thiosulfates using a detector which is an indicator of intestinal inflammation derived from the Shevchenella marine. They planned to produce a pill which contained the E.coli that would produce a chromo-protein to change feces’ color. The excrement color will be affected by the pill, as an indicator of IBD. However, there were few problems about the design. 1) Even though E.coli is probiotic, this method may have certain biological risksS. 2) the promoter leaks seriously which caused the result inconspicuous. 3) this pill can only qualitatively test thiosulfate. Based on this experiment, our project has done a lot of improvements and promotions this year. Compared to the 2017 experiment, our 2019 iGEM team is dedicated to separate the detector from the reporter and do quantitative analysis. We would collect bacteria and give them to the institution or professionals, allowing them to give a more accurate assessment of intestinal inflammation.

Second, in order to let the cell receive the signal and record it on the DNA sequence, we planned to use the SCRIBE system (synthetic cellular recorders integrating biological events). SCRIBE is constructed by three systems— MSR-MSD, RTEC86, BETA Recombinase (Fahim, F & Timothy K. Lu 2014). This recording system uses reverse transcription and recombination process to transform the external signal into a genome mutation signal. Also, we researched the CAMERA system (CRISPR-mediated analog multi-event recording apparatus), which can record biological, chemic or environmental signals. When receiving certain stimulus signals, cells will express Cas nuclease and gRNA to change the sequence of reported gene. Once the sgRNA successfully combines with corresponding DNA target site, certain Cas nuclease will be able to edit the target sequence. Subsequently, it will gradually repair the damaged GFP. We envisioned to use these systems to edit the stop codons to fix the target DNA.

Third, for the part of reporting genes, we found a standard way to quantify the lightness of GFP. Before repairing, the GFP is damaged which cannot fluoresce, yet after repairing, GFP can fluoresce in different degree. After researching a reference, we found a reliable stable and standard protocol of measuring gene expression to test the proportion of modified GFP.

Under this framework, we randomly identified different point mutation in GFP, then experimented and identified a GFP that had the relative low pre-repair expression and a significant difference after repair.

Finally, in terms of hardware, we planned to design a capsule. This capsule needs to keep the digestive enzyme molecules out, make sure the bacteria inside cannot leave the capsule and allow small molecules to penetrate the capsule’s membrane. Ergo, we found a semipermeable membrane to fulfill the requests. In addition, we plan to add a magnet on the top of the capsule, allowing the patient to find the capsule easily, and the design of the capsule makes our recycling operation more convenient.