Team:XMU-China/Improve
This year, we have improved 2 previous projects. The one is the project of WHU-China 2014, and another one is the project of XMU-China 2016. And we have improved 5 BioBricks, BBa_K1334005, BBa_K1334015, BBa_K1334016, BBa_K1334017 and BBa_K1960103.
----* Improvement of WHU-China 2014 *----In the project of WHU-China 2014, they made attempt to deal with formaldehyde pollution, but they ended with a failure. This year we conducted a new system on the basis of WHU-China's effort, redesigned and improved their project with our methods.
I. Improvement of the Street Cleaner System
In the project of WHU-China in iGEM 2014, they tried to absorb formaldehyde using The Street Cleaner System, which contains formate dehydrogenase (BBa_K1334016) and formaldehyde dehydrogenase (BBa_K1334017), but these two genes did not express.
Figure 1. The Street Cleaner System designed by WHU-China 2014
This year, we redesigned the Street Cleaner System by replacing the PADH and FADH with another coding sequence, which codes formaldehyde dismutase, BBa_K2310900. This enzyme enjoys high catalysis activity of translating formaldehyde into carbon dioxide and methanol. Although XMU-China 2016 also designed another BioBrick, BBa_K1960103, which also codes formaldehyde dismutase, there are still some problems with that part. The sequence of BBa_K1960103 is from Halomonas sp.JC-5B, a formaldehyde tolerance bacteria in Pacific Ocean, and this kind of bacteria is different from E.coli, so the efficiency of expression of BBa_K1960103 is very low. To solve this problem, a codon optimization should be needed.
We redesigned the sequence of BBa_K1960103 with a codon optimization, and synthesized the new gene by IDT. The redesigned version of formaldehyde dismutase is BBa_K2310900 .
After that, we cloned these two genes into plasmid pET-28a with a His-tag, induced with IPTG to test the efficiency of expression with a precise quantification.
Figure 2. SDS-PAGE for BBa_K1960103 and BBa_K2310900
(Both of them with a His-tag is about 50kDa)
The SDS-PAGE shows that our improved version of formaldehyde dismutase, BBa_K2310900, has a much higher efficiency of expression compared with BBa_K1960103.
After testing the efficiency of expression, we also tested the catalytic effect of these two enzymes. After a 6 hours inducing with IPTG, we resuspend the bacteria with corresponding plasmid with formaldehyde solution, and incubated at 28℃, assay the content of formaldehyde once an hour.
Figure 3. The enzyme activity assay of BBa_K1960103 and BBa_K2310900
The result shows that our BBa_K2310900 works well.
II. Improvement of the coloration system
To detect formaldehyde in the environment, WHU-China 2014 designed a coloration system. They designed a promoter, BBa_K1334002, which can be activated by formaldehyde, and then add a fluorescent protein downstream. In this way, people can know whether there is formaldehyde or not from the color change.
Figure 4. The coloration system designed by WHU-China 2014
This system designed by WHU-China 2014 has a shortcoming: the expression of GFP is controlled by the promoter BBa_K1334002, and the strength of this promoter may be very low, so we cannot get enough fluorescence intensity that is easy to be detected. To solve this problem, we redesigned this system with our Bio-amplifier.
Figure 5. The redesigned coloration system
As we all know, T7 promoter is a very strong promoter while T7 RNA polymerase exist, so we designed a Bio-amplifier based on this theory in our main project. To improve the project of WHU-China 2014, we used our bio-amplifier to enhance the strength of BBa_K1334002 as Figure 5 shows.
Figure 6. The fluorescence intensity of two coloration systems (the concentration of formaldehyde is 0.1mM）
As Figure 6 shows, our improved coloration system has a stronger fluorescence intensity, about 1.4 times stronger than the coloration system designed by WHU-China 2014.
In the project of XMU-China 2016, they designed a circuit to detect and kill antibiotic-resistant bacteria, and the most attracting part of their project is their toggle switch, which can bring a delayed effect with a 3 hours delay.
Figure 7. The designed circuit of XMU-China 2016
But in their project, they only finished the experiment part of their project without modeling. After learning about their project, improved their project by modeling their circuit.
Because XMU-China 2016 did not get enough data to complete their modeling, we have to look for more data from their references[1] and repeat their work to complete and test the model.
I. The relationship between the concentration of AHL and the fluorescence intensity of GFP
To get a formula which can show the relationship between the concentration of AHL and the fluorescence intensity of GFP, we fitted their curve using GraphPad PRISM software.
FI: fluorescence intensity
AHL: concentration of AHL
II. The function of the circuit
The models were based on ordinary differential equations that captured the activation and repression of protein synthesis. The intracellular species included GFP (G), LacI (L), cI (C), LuxR/AHL complex (R), AHL (A) and a fixed concentration of LuxR. The following equations were used:
[1] Subhayu Basu et al., A synthetic multicellular system for programmed pattern formation, Nature, 2005, 434, 1130-1134.
[2] Yates, E. A. et al., N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa, Infect. Immun., 2002, 70, 5635–5646.
Xiamen University, Fujian, China
No. 422, Siming South Road, Xiamen, Fujian, P. R. China 361005
