Collaboration
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.
Mentoring with UCCKE
The Hong Kong UCCKE team aims to create biobricks that detect and control gout by degrading uric acid and transport it. The Hong Kong UCCKE team presented their idea to us and we proposed that they can run an assay with plate reader measuring the fluorescence signal of the detection module, and incubating transformed E. coli in LB medium with saturated uric acid over night for the assay of the degradation module. Detailed protocols can be viewed below.
We have visited each others lab and presented our project in July, and we have suggested them to amplify the synthesized DNA from IDT for stock and use screening method other than restriction digestion(e.g TA clone) as the insert and vector have similar size. We also pointed the need of negative and positive controls in their assays.
Apart from providing advice and analysing the results, we also provided instrument for the standard cloning of their biobricks. As one of the secondary schools with advanced lab settings, they still lacks important devices like -80C fridges for storing competent cell. Thus we helped them to clone biobricks in parallel. We also helped preparing the sequencing of their plasmids and perform the uric acid assay through plate reader for their referece.
In return, the Hong Kong UCCKE team realised our hardware detection kit by translating our blueprint into 3d model and printed it for us. They also provided us suggestions on the improvement of the prototype regarding 3d printing production. The resulting product is as shown in the following figures.
Collaboration with HKUST
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.
The test result of the characteriation of 6 biobricks provided by Hong Kong HKUST team. 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.
Protocols for collaborations:
UCCKE:
Uric acid assay
The protocol is designed and provided by us.
Materials:
Project300 part in DH5a E coli
LB+ Chloramphenicol
Uric acid
96 well plate+ ClarioSTAR plate reader
Procedures:
1.Transform 300 plasmid into Dh5a E coli cell
2.Pick a clone for the 300 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.
HKUST:
The protocol is provided by HKUST here.
