iGEM 2021 — Team THIS-China

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# Team:THIS-China/Notebook

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THIS-China - Notebook

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# Weeks 1 &  2 (May 29 – June 12)

1. Preparation of experiment, getting familiar with the lab techniques and lab apparatus.
2. Start construction of circuit, preparing individual parts of circuit.

# Week 3 (June 13 – 19)

1. Construction of the circuit

comE, comD, and cotA in vector (PDG-148). **Important issues:** results from the first experiment session of the third week show that the construction of our genetic circuit has not been successful. We therefore changed our strategy to first combining the comE and comD with the vector.

2. Construct Circuit #1

comE and comD in vector (PDG-148)

# Week 4 (June 20 – 26)

1. Construction of circuit #1 using Gibson assembly

**Important issues:** The results from gel electrophoresis show the correct base pair numbers. But the sequencing test obtained show different results from the expected design of our circuit. We therefore decided to switch and use the traditional restriction enzyme site cutting protocol.

Redesigned the primers for our new protocol.

2. Construct Circuit #1

Using restriction enzymes instead of Gibson assembly for the genes comE and comD and the plasmid PDG-148.

# Week 5 (June 27 – July 3)

1. Successful construction of circuit #1
2. Constructed circuit #2

Restriction enzymes sites BamHI-HF and Xhol were used to cut and connect cotA in plasmid.

3. Verification of sequencing through TIAN prep Mini Plasmid Kit and Nanovue.
4. Gel electrophoresis show successful construction of circuit #2.

# Week 6 (July 4 – 11)

1. Thought of substituting cotA protein with other direct color making chromoproteins.
2. PCR amplification of different color chromoproteins, including pink, purple, green, yellow, and blue.
3. Transform them to plasmids with comE and comD into E.coli and conduct transformation to Bacillus subtilis 168.

**Important issues:** Bacillus subtilis 168 is working badly because they are extremely hard to cultivate. Therefore, according to research from the group STPZ-shenzhen, we decide to use E.coli.

# Week 7 (July 12-17)

1. The competent cell DH5alpha demonstrated an effective completion of the circuit, and the trial with purple chromoprotein showed a faint color. To aim for better expression of the colors, we tried DH5a with different concentration levels, hopefully observing a pattern.
2. Transformation and growth of multiple competent cells including Rosetta, DH5a, and Bu21-pog148. Observe expressed colors. Results show that only purple had a faint color.

# Week 8 (July 18 – 24)

1. Insert plasmids with yellow, green, and pink chromoprotein genes again into bacillus. Attempt for expression of fluorescent gene and observe effectiveness. Tested them with negative control.

**Important issues:** First, the other colored chromoprotein is not successfully expressing (the color is not dark enough for it to be easily seen). Second, the promotor nlmc is leaking when these chromoproteins are expressed because they are too strong. We decided not to use chromoprotein anymore for substitution.

# Week 9 (July 25 – 31)

1. Transformation and growth of bacteria with circuits #1 and #2 together (putting comE, comD, and cotA together).
2. Gel electrophoresis passed, but the protein electrophoresis for comD and comE did not appear, which means that these two proteins did not express.

**Important issue:** to solve the problem of protein not expressing successfully, we tried to use two plasmids with constructed circuit to increase the chances of expression for comE and comD. One plasmid with comE and cotA gene and the other with comD separately.

# Week 10 (August 1 – 7)

1. Growth of cell with pET-21 and pET-28 vectors (substituting the pdg-148) with different gene and resistance.
2. Vector pET-21 and gene comE and cotA is combined then transformed to the ecoli-BL21.
3. The transformation is successful and passes the gel.

# Week 11 (August 8 – 14)

1. Vector pET-28 and gene comD is combined and PCR of plasmid then transformed into competent cell ecoli-BL21.
2. The transformation is successful and passes the gel.

# Week 12 (August 15 – 21)

1. Transformation of the 2 plasmids together into one E.coli.
2. Went through protein gel electrophoresis and found out that the protein expressed properly.

# Week 13 (August 22 – 28)

1. Do different groups to check the whole circuit by adding RR color for it to degrade.

Five groups each with the following: only cotA, cotA and IPTG, comDE and cotA, comDE and cotA and IPTG, and comDE and cotA and IPTG and CSP-1.

2. Found out that the cotA degradation protein works separately.

**Important issue:** CSP cannot be detected by the comDE system because they do not express. Solution is proposed in the Parts and Future Goals.

# Week 14 (August 29 – September 5)

1. Test the penetrability of CSP-1 in 5KD filters in the presence of bacteria with sizes 0.8μm~500μm.

# Week 15 (September 26– October 1)

1. Conduct experiments to check whether the comD protein is correctly on the membrane or not.

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# Team:THIS-China/Engineering

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THIS-China

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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

![](https://static.igem.org/mediawiki/2021/f/f9/T--THIS-China--9Picture1.png)

**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

![](https://static.igem.org/mediawiki/2021/3/39/T--THIS-China--9Picture2.png)

**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.

![](https://static.igem.org/mediawiki/2021/f/f4/T--THIS-China--9Picture3.png)

**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.

![](https://static.igem.org/mediawiki/2021/1/14/T--THIS-China--9Picture4.png)

**Figure 4**: Two plasmids we used.

![](https://static.igem.org/mediawiki/2021/a/aa/T--THIS-China--9Picture5.png)

**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.

![](https://static.igem.org/mediawiki/2021/2/27/T--THIS-China--9Picture6.png)

**Figure 6**: Data for color degradation.

![](https://static.igem.org/mediawiki/2021/c/cf/T--THIS-China--9Picture7.png)

**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

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# Team:THIS-China/Experiments

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# Team:THIS-China/Results