Overview

     We added new information learned from literature and the experimental characterization to the previous parts (BBa_K143057, BBa_K322921, and BBa_K1054002). Furthermore, we have documented the optimized method to transfer Bacillus subtilis with the help of PI Xie’s lab. Meanwhile, we built two models to validate our feasibility of our overall design and offer a method to depict the concentration change of chemical substances, which may be useful to future iGEMers. In addition, we manufactured a portable dual port filter for bacteria activation to assist the wet experiment and designed a device to simulate the fluid and intestinal peristalsis in the intestinal tract, so as to determine the final form of drug external package.
     All of these may be helpful and useful to other teams, and we hope they will make some contributions to the iGEM community.

Wet-lab

Pxyl-SpoVG RBS (BBa_K143057)

     In our project, we search for an inducible promoter in Bacillus subtilis to express our SAMe transporter, and we found this part. However, the information is not enough to help us to describe such part, so we add some literature information and some our experiment results here. We used such expression to express our SAMe transporter opPet8p. When we used such part, we found that the inhibition of XylR could be not strong enough. Without xylose induction, we can also found green fluorescent signal (Fig. 1). So to reduce the leakage of downstream protein expression, we planed to perform directed evolution.

Figure. 1 Fluorescent Microscopy of B. subtilis with pBE-XylR-Pxyl-opPet8p::sfGFP without xylose

    According to literature, this xylose-induced promoter can be engineered for different application. It can be used as a genome engineering method[1], and it can be used to construct a xylose-inducible Bacillus subtilis integration[2], and so on.

SacB (BBa_K322921)

    This is a sucrose lethal gene that encodes the enzyme levosucrase, which catalyzes the production of high molecular weight fructose polymers called Levans. Given that heterologous expression of SacB is lethal in the presence of sucrose in many Gram-negative bacteria, SacB is widely used as a genetic engineering tool.
    In our project of directed evolution , we added SacB in our constructed vector as a counter-selection element , and we collected some related information from the past papers to verify our experiment [3,4,5,6].

Figure 2.1. SacB addition in our constructed vector , which serves as a counter-selection element.

Figure 2.2 In the process of counter-selection,we add sucrose on the surface of solid culture medium . (A): No colonies are found on the medium supplemented with sucrose;(B):There are many colonies on the medium without added sucrose.

PBSX holin (BBa_K1054002)

    PBSX holin is a small membrane protein in double-stranded DNA phages for efficient host lysis. Previous research has found that Holins are the gatekeepers of the lysis process, possessing an intriguing ability to be triggered, at a precise time point, to form large holes in the cytoplasmic membrane of phage-infected bacteria. ([7] Mart Krupovič, Dennis H. Bamford. 2008)

Figure 3.1 The ‘death raft’ model for holin lesion formation. (Mart Krupovič, Dennis H. Bamford. 2008) In this research, a ‘death raft’ model is used to describe holin lesion formation. The process includes accumulation and oligomerization of holins in the CM, formation of the ‘death raft’, the opening of an aqueous channel and formation of the holin lesion. (Figure 3.1)

    In this research, a ‘death raft’ model is used to describe holin lesion formation. The process includes accumulation and oligomerization of holins in the CM, formation of the ‘death raft’, the opening of an aqueous channel and formation of the holin lesion. (Figure 3.1)
    After understanding the functional mechanism of holin, we predicted holin’s structure by PolyPhobius prediction. It shows that PBSX holin has two transmembrane regions. (Figure 3.2)

Figure 3.2 The prediction of the transmembrane domain of PBSX holin.

    Inspired by the properties of holin mentioned above, we think holin is an effective and secure choice for our kill switch, for it will cause the cleavage of the engineered bacteria only, releasing no toxin in the gut or environment. We induced the expression of PBSX holin in E. coli at the log phase and measured OD600. The result shows the ability of holin to inhibit the growth of bacteria to some extent. (Figure 3.3)

Figure 3.3: Induction of the expression of PBSX holin in E. coli.
A. Induction on a culture plate.
B. Change in turbidity.

Document the method to transfer Bacillus subtilis

    It’s reported that gram-positive engineered bacteria are difficult to transfer, since they have a thick cell wall and complicated genetic background. Here we have documented two optimized ways and notes to transfer Bacillus subtilis to increase the transformation efficiency. We believe such protocols will help other teams work on gram-positive bacteria instead of being limited to Escherichia coli DH5α.

Reference

[1]Jeong DE, Park SH, Pan JG, Kim EJ, Choi SK. Genome engineering using a synthetic gene circuit in Bacillus subtilis. Nucleic Acids Res. 2015 Mar 31;43(6):e42. doi: 10.1093/nar/gku1380. Epub 2014 Dec 30. PMID: 25552415; PMCID: PMC4381049.
[2]Kim L, Mogk A, Schumann W. A xylose-inducible Bacillus subtilis integration vector and its application. Gene. 1996 Nov 28;181(1-2):71-6. doi: 10.1016/s0378-1119(96)00466-0. PMID: 8973310.
[3]Ambrosis N, Fernández J, Sisti F. Counter-Selection Method for Markerless Allelic Exchange in Bordetella bronchiseptica Based on sacB Gene From Bacillus subtilis. Curr Protoc Microbiol. 2020 Dec;59(1):e125.
[4]Chen W, Li Y, Wu G, Zhao L, Lu L, Wang P, Zhou J, Cao C, Li S. Simple and efficient genome recombineering using kil counter-selection in Escherichia coli. J Biotechnol. 2019 Mar 20;294:58-66.
[5]Logue CA, Peak IR, Beacham IR. Facile construction of unmarked deletion mutants in Burkholderia pseudomallei using sacB counter-selection in sucrose-resistant and sucrose-sensitive isolates. J Microbiol Methods. 2009 Mar;76(3):320-3.
[6]Tan Y, Xu D, Li Y, Wang X. Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum. Plasmid. 2012 Jan;67(1):44-52.
[7] Mart Krupovicˇ and Dennis H. Bamford. Holin of bacteriophage lambda: structural insights into a membrane lesion. Molecular Microbiology(2008)69(4), 781–783.

Hardware

Portable Dual Port Filter

    We used 3D printing technology to make a dual-port filter. It can help us activate bacteria and achieve solid-liquid separation. This device will facilitate the subsequent wet experiments.

     We also tested the feasibility of the device and achieved the expected results, that was, the variation of the bacterial concentration after passing through this instrument. The experimental results are as follows:

Enteral Drug Simulation Device

    We used micro flow pump and synchronous motor to simulate the fluid and intestinal peristalsis in the intestinal tract, so as to determine which chemical substance could be used as the external package of drugs. In this way, drugs can be absorbed better in the intestinal tract.

     Finally, we tested the dissolution rate of different chemicals at different pH, and found that carboxymethyl cellulose can satisfy the rapid dissolution in the intestinal tract and keep stable in the stomach.

Model

PEST Analysis

    We are excited to share our modeling ideas with future iGEMers, in hope that our methods can do a little favor. Our first model based on SimBiology validates the feasibility of our overall design pathway. Other teams utilizing complex biological pathways can likewise use it to perform preliminary validation of their designs. Our second model provides a method and idea to depict the concentration changes of some chemical substances, which may be helpful and useful to the future iGEMers. Moreover, we use some technique to display final results dynamically, which helps judges input relevant parameters and obtain simulation results more obviously. This idea might inspire other teams.

HP

    In order to gain more knowledge about synthetic biology and make suitable advice on our project, we have done a lot in Human Practice. First, by interviewing professors, enterprise stakeholders (for example, the shop assistants), etc., we aimed our project to solve the problems that people are troubled with and concerned about.

    Moreover, in education part, we expected others not only to know depression scientifically, but to be familiar with synthetic biology as well. In order to better understand the patients' needs, we have conducted several communications with DO & GO, an online community for people with depression. And in this year, we are the host of Central China iGEM Communication Huazhong Meetup, which helps to utilize synthetic biology and our innovations to benefit mankind and the society.

     Last but not least, to build a large family of synthetic biology, we have conducted rich communications, which deepen our understanding of synthetic biology and our own project significantly.

    Briefly, our team this year fulfilled the goals of inclusivity and education in a reciprocal way.

Entrepreneurship

    It is our pleasure to exchange our project with Bluepha, BGI, and universities like NJMU, and obtain affirmation and support from them. They provided data to help us complete the market research and analysis of antidepressant drugs in the Chinese market, and make a complete business plan for our project, so as to comprehensively consider and design the commercial promotion and application prospect of our products in the future.