Collaborations

Collaboration with university team

Collaboration with NUS Singapore Team-A

Our team did not get desirable results when probes are expressed in E. coli BL21 (DE3). While Team NUS Singapore-A initially wanted to explore using RNA aptamers as a reporter system for the stress promoter htpG1, they had difficulties in cloning the construct for months. When they discovered that we are working on aptamer and the expression of it in E. coli, their team leader, Nanda, has a small meet-up with our wet lab team members in Hong Kong. We saw that our projects are closely related and there is an opportunity for collaboration.

*Nanda (far left) with the members from CUHK iGEM team*

After the discussion, we found out that their team has BL21 Star (DE3), a RNase-knockout strain of E. coli. As we expected see stronger signals from expressing the fluoescent RNA probes, we asked for this strain from them, which they kindly delivered it to us in the form of dried filter paper. We made competent cells from it and performed the assay again.

On the other hand, NUSGEM needed help on RNA aptamer constructions. While de novo gene synthesis or PCR from plasmids did not work for them, we have succeeded in cloning using oligo PCR, a design that allows the partially overlapping oligos to extend and form the desired gene fragment. We attached the Spinach aptamers to either a constitutive lpp promoter as a positive control, or a htpG1 promoter for their assay. Both teams agreed on constructing the constructs from both teams simultaneously, using the set of primers which our team has designed. In the end, their cloning was finally successful and is documented in the parts registry (BBa_K2819010).

CUHK and NUSGEM results — *Gel Electrophoresis of PhtpG1-RNA aptamers*

As NUSGEM has also constructed the lpp-aptamers, we asked for their help to measure the fluorescence intensity of iSpinach-D5 under different assay temperatures. It is found that iSpinach-D5 is brighter and more temperature-resistant than the previous biobrick, Spinach2.1.

Lastly, NUSGEM helped spreading our survey regarding influenza-flu differentiation for gathering responses in Singapore.

Cooperation with team Uchile

Both our team and team Uchile are trying to make use of aptamer to simplify the detection process. Thus we get in touch with each other to discuss our common problems and give feedback to each other.

Team Hong_Kong CUHK’s feedback for team Uchile:

Maybe in the presentation you could give more details on why you choose to detect this toxin instead of other toxin?
For the project, have you compared your accuracy with the current widely-used method?
In the results part, could you put some labels on the rows and columns on your results page because we are a little confused about what is inside the samples.
For the hardware part, we are still quite unclear about the design of your hardware, would you mind offering some further details on that?

Team Uchile’s feedback for team Hong_Kong-CUHK

biosecurity: Will tracer be reusable? is it possible that with the misuse of the hardware, contamination can be generated? How will you proceed in case the hardware is discarded?
Answer: Tracer is reusable and contamination will not be generated if people follow the user guide.Tracer is intended for the general public, if people are diagnosed with influenza, then we will suggest the user to give Tracer to the local hospital/clinic to disinfect before it is reused.
In the results part, could you put some labels on the rows and columns on your results page because we are a little confused about what is inside the samples.
Answer: The refractive index of PLA is as low as 3%, which can prevent the diagnostics results from the influence of the outside environment. Moreover, with a melting point of around 160 °C, PLA provides great heat stability. Also, PLA is an environmentally-friendly material as it is biodegradable.
The proteins of the influenza present variable proteins. You have to be constantly making new vaccines. with his method it is said that it is easy to produce new sequences but will it be on the same level with the mutation rate?
Spinach is destabilized when the length P1 <5. if it is 5pb (short), how can you be sure that it binds specifically to the virus and not something else?

Changes we made to our project after meeting with Uchile:

We considered the problem of how to deal with the samples after testing, which leads us to change from using cuvette to eppendorf, since eppendorf is easier to heat.

Read our meeting note Team Uchile Homepage

Collaboration with Highschool Teams

Collaboration with Team Hong Kong JSS

Team Hong Kong JSS is a joint highschool team. We collaborated with two of the schools.

Yan Oi Tong Tin Ka Ping Secondary School

1 teacher and 3 students from the school came to our lab SC364, and we taught some synthetic biology techniques to them. They performed transformation of E.coli TOP10 and spreaded plates under our supervision. They helped us to spread our survey regarding cold-influenza distinction in their school and we have collected the opinions from a younger audience.

United Christian College

4 secondary school students from the school came to our lab SC364. We provided competent cells and plates for them to carry out their Interlab experiment. We also provided our plate reader BMG CLARIOStar for them to measure absorbance and fluorescence for their samples. In addition, they helped us to spread our survey regarding cold-influenza distinction in their school and we have collected the opinions from a younger audience.

Team:Hong Kong-CUHK/Collaborations