Overview
This year, our team submitted some documents from experiments to several different parts. In addition, the results of our mutagenesis experiments through the RNA thermometer present troubleshooting, all of which are detailed below. In the aspect of human practice, our team made a probiotics handbook with LZU-HS-PRO-A to help people learn how to buy and use probiotics.
Characterize Existing Parts
We completed the experimental characterization of the parts (BBa_K3733043 and BBa_K3838613) and added some information about them.
Butyric acid validation
From the literature, we learned that butyrate not only has anti-inflammatory effects in the intestine[1], but also enhances the intestinal barrier, reduces intestinal permeability, circulating LPS, and systemic inflammation, thereby indirectly inhibiting the formation of atherosclerosis[2]. The thioesterase expressed by the Tes4 gene hydrolyzes the thioester bond of the acyl-S-acyl proteins, promoting the release of short-chain fatty acids, including butyric acid[3].
To verify that thioesterase was present and functional, the supernatant was subjected to gas chromatography, which confirmed the production of butyric acid[3].
Explanation of two peaks in the second picture: we suspect that Escherichia coli BL21 (DE3) (with His-tag) itself will produce a substance with properties similar to butyric acid. According to the gas chromatography results of the butyric acid standard solution, it can be proved that the peak time located between 9.6min and 9.7min shows our target product, butyric acid (Figure 1)
.
Suicide Module
To ensure the safety of the engineered bacteria , we designed a suicide module, killing the strains, which leak outside the body may contaminating the environment. Considering the temperature-differences between the human gut and the external environment, we referenced the temperature-sensitive suicide module of HZAU-China 2021 (BBa_K3733043) and introduced this circuit of cold-induced suicide system.
In this suicide module, we designed a heat-repressible RNA thermometer with a toxin protein followed. The former senses the ambient temperature and plays a role in controlling the switch close and open which influence the follow-up pathway, while the latter is able to express the toxin protein HepT, acting as a RNase, which degrades mRNA at the transcriptional level and ultimately leads to cell death. Through such design, we ensure the suicide of the engineered bacteria in vitro, furthest reducing the environmental pollution.
The circuits of the schemes and brief mechanism of Suicide module is shown below.
Troubleshooting
Process and result
Based on the elements of HZAU-China 2021 (BBa_K3733043) of the suicide plate of HZAU-iGEM, we first recombined the RNA thermometer vector and eGFP fragment homologous into E. coli DH5α competent cells and Colony PCR was used to screen the correct homologous recombinant bacteria. Then, the mutation was purified by gel recovery and transformed into E. coli DH5α. However, the control and mutant groups did not glow at any temperature.
Speculation and Prospect
Our guess is that some other part of the constructed plasmid is mutated so that the gene is not properly expressed. But for reasons of time we have not been able to ascertain exactly why. Finally, we suggest that future teams test the whole plasmid or carefully select this part as your temperature switch.
Probiotics Handbook
To help people use probiotics properly, HZAU-China and LZU-HS-PRO-A have made a probiotics handbook to help people buy and use probiotics. The manual introduces the functions and principles of different kinds of probiotics in detail, which can be used as a reference when people buy probiotic products in their daily life (Figure 3).
In human practice part, we communicated with ZJUT-China in the intestinal information platform. They asked some questions about oral and gut health and probiotics, and we recommended after them and discussed with them.