Every iGEM team have its strength and weaknesses. And every project have flaws that could not be seen in a single perspective. Through in-depth collaborations, we can complement each other’s weakness with our strength, and refine our project by evaluating one another.
The Hong Kong UCCKE team aims to create biobricks that detect and degrade uric acid in order to control gout disease. We mentored the team by providing advice, protocols and also helping them to do experimental work. In the beginning of their project, they had difficulty in designing their experiment, especially when designing the assay for characterizing their biobricks. We helped them to construct an assay for measuring the fluorescence signal of their detection module. The assay can be done by incubating transformed E. coli in LB medium with saturated uric acid overnight followed by measuring fluorescent signal in plate reader. The detailed protocols can be viewed below. When they started doing their wet lab, they encountered difficulty in cloning their biobricks. To help them solve the problem, we have provided many advice. For example, to deal with the low insert concentration problem, we suggested them to amplify the synthesized DNA before doing restriction cut. We also suggested them to do restriction map before sequencing. We emphasized them the importance of negative and positive controls when troubleshooting problem of cloning and assaying. At the final stage of the project, we helped them to clone biobricks in parallel. We also helped them to sequence their plasmids and analyze sequencing result. Luckily, they constructed their biobricks on time. After having the biobricks, we helped them to perform the uric acid assay because they do not have plate reader in their school. In return, the Hong Kong UCCKE team helped us to 3D- print our hardware detection kit. They also provided us suggestions on the improvement of the prototype. They helped us a lot because we do have any knowledge in product designing and 3D printing previously.
The Hong Kong HKUST team aspire to create a safety switch that would not compromise the functionality of original biobrick, but able to revert the transformant back to wild-type with time. We helped HKUST team’s progress by providing them a Cython-implemented Python interface. It enables them to use the ViennaRNA package functions(written in C language) in Python interface, so they can easily model the dissociation constant for the antisense mRNA and transcript mRNA.
We also help characterizing their biobricks by running an GFP assay, detailed protocol and results attached below. In return, they provided us two Python scripts for our modelling of free energies of opened and closed toehold switches, by plotting ODE-based model and stochastic model using Euler’s method and Gillespie algorithm respectively. This facilitates our screening of optimal toehold switches in our program.
Below showed the test result of the characteriation of 6 biobricks provided by Hong Kong HKUST team. (Detailed interpretation of data is in their wiki)The biobricks were incubated with varying concentrations of AHT and measured the GFP fluorescence with COSTAR plate reader. Only pSB1C3-BBa_F2620-C0261-E0240-R0063-Anti2 shows a positive trend with increasing AHT concentration. In contrast, Hong Kong HKUST team's own results show a positive trend with pSB1C3_BBa_T9002.
Uric acid assay
(The protocol is designed and provided by us)
Detection part in DH5a E coli
96 well plate+ ClarioSTAR plate reader
1.Transform 300ng plasmid into Dh5a E coli cell
2.Pick a clone for the 300ng plasmid into 5ml LB with Chloramphenicol
3.Prepare saturated uric acid solution with LB solution and perform 1:1 serial dilution to make 5 different concentrations of uric acid in LB.
4.Inoculate 2ml of LB into 250 ml of LB with Chloramphenicol and varing concentration of uric acid in 37C overnight.
5.Measure the fluorescence of GFP(absoprtion:583nm emittion:607nm) of 200μl of biobrick by ClarioSTAR plate reader.
The protocol is provided by HKUST here.