Team:THIS-China/Engineering

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Design

Our experiment aims to build a sensor cell by implementing a genetic circuit into the host cell of E. coli or Bacillus subtilis. The CSP will activate the membrane protein comD and the protein comE, which will phosphorylate and activate the cotA enzyme gene to cause color degradation. The final result should be visible to the human eye. After the CSP combines with comD and comE and activates the cotA gene, the red color should be degraded to a yellow color. We propose to use two types of host cells, E. coli DH5-alpha and Bacillus Subtilis, for the transformation of the system

Figure 1: Our product's genetic circuit.

Build

We have two potential methods of building our system in our host cell. In the E. coli cell, we can use the Gibson Assembly because it is an efficient way to connect the PCR product to plasmids (backbone PDG-148 and genes comE, comD, and cotA). A second way is to use the restriction enzymes BamHI-HF and Xhol to construct the system.

Test

With using various PCR and transformation methods, we first tried to use Gibson Assembly to connect the PCR product of comD, comE and cotA with the plasmid. After this, we transformed it into the E. coli cell. After encubating this in a 37-degree environment, no bacteria were grown

Figure 2: Gibson Assembly design.

Learn

There are usually four reasons for unsuccessful transformation: 1) primers could not be aligned and so they did not align with our plasmid pDG-148, 2) the thermo-cycling setting was not suitable for the genes, 3) the overhangs are not specific and connect to random sites, or 4) the plasmid is not compatible with the system. After going through our process to narrow down the possibilities, we conclude that the problem may be due to unspecific overhangs or an E. coli type that was not suitable for the transformation.

Redesign/Rebuild

After not succeeding in the Gibson cloning method, we used the traditional method of using restriction enzymes to connect the parts. This method is different from Gibson assembly in that the DNA fragments are connected multiple times using different restriction enzymes, whereas they are all combined together at once in the Gibson Assembly. In this part of the experiment, we decided to try out E. coli DH5-alpha and BL21 for the host cell to make sure that the E. coli will be suitable for the transformation.

Test

After running an overlap PCR for cotA and DNA purification, we connected the three segments of cotA and (PDG-148, comE, comD) with the restriction enzymes BamH-HF and Xhol. Then we connected these two segments with T4 DNA ligase buffer to create the full genetic circuit. Finally, we transformed this system into E. coli DH5-alpha and E. coli BL21.

Figure 3: Restriction enzymes design.

Learn

We tried this restriction enzyme method and transformed it into two types of E. coli cells, DH5-alpha and BL21. The ones in the DH5-alpha did not grow, but it worked in BL21. BL21 Competent E. coli is a widely used non-T7 expression E. coli strain and is suitable for transformation and protein expression. DH5-alpha Chemically Competent E. coli cells are suitable for high efficiency transformation in a wide variety of routine applications, such as plasmid isolation, cloning, and subcloning. So, it is possible that some components of our plasmid and gene circuit were not suitable for the DH5-alpha cell. We also tried it two times, first in the PDG-148 plasmid and incorporated into the whole system, which did not grow, so we put it into two plasmids PET-21 and PET-28 for transformation. Putting comD and comE in PET-21 and cotA in PET-28 E. coli cell was successful and the bacteria grew.

Figure 4: Two plasmids we used.

Figure 5: Incubate E.coli BL21.

We tested the degradation coloring with five groups, each with: only cotA, cotA + IPTG, comD + comE + cotA, comD + comE + cotA + IPTG, and comD + comE + cotA + IPTG + CSP-1. We found out that the cotA degradation protein works by its own, but the others did not work and it was not detected by the systems containing comE and comD.

Figure 6: Data for color degradation.

Figure 7: Color degradation.

References

https://www.neb.com/products/c2530-bl21-competent-e-coli

goldbio.com/product/14438/dh5-alpha-chemically-competent-e-coli-cells