Team:Hong Kong HKUST/Collaborations
Collaboration with HK_GTC
This year, we collaborated with a high school team, GT College, on many fronts:
Transformation and miniprep
The team wanted to characterize multiple RBSs to fulfil the requirements of characterizing an old part. They wanted to clone different RBSs to BBa_E0040 GFP by PCR overhangs. However, they did not succeed in PCR. Team members of the GTC team sought help from us by asking us for a plasmid that contains BBa_E0040 GFP. We transformed a psB1C3 plasmid with insert BBa_K608002-BBa_I13401, and performed plasmid extraction on one of the colonies. With help from us, the HK_GTC team successfullu cloned their desired product and used the construct for old part characterization.
 Our team menber passed plasmid and transformed colonies to a team member of HK_GTC. |
 Tramformed GFP colonies shows green as expected. |
The GTC team also asked for an inventory of the 2018 HKUST team, BBa_K2764002, as an old part for characterization.
Troubleshooting
We also conducted a troubleshooting meeting with GT College to discuss our projects. During the meeting, we exchanged ideas on our project design and helped them troubleshoot their PETase expression. Their cell lysate SDS-PAGE was not able to indicate the positive expression of PETase. We therefore suggested them to carry out western blotting or Ni-NTA column purification before running SDS-PAGE, and taught them about these processes.
Modelling
Analogous to how the Collins toggle switch was modelled, our team also created a generic model that could be made to fit the different potential applications of our circuit. Thus, we wanted to test the effect of changing the constants that might be brought about by the customization of the future user using evaluation of elasticity. For their part in the collaboration, the GTC team agreed to test our modeling for us using different sets of constants and inputs to test for the elasticity.
We sent them a set of python code for our model, along with a list of constants and inputs to be tested. They managed to generate an array of data, which helped us to understand the behavior of the circuit with respect to changes in constants and inputs. Their data also further helped us to improve our model.
Mentoring the Hong_Kong_UCCKE iGEM team
We also mentored a high school team, UCCKE, by giving them wet lab training, modelling workshop, and helped them in cloning and transformation.
Wet lab training
Our team member Owen explained some common cloning strategies and RFC10 standard assembly (using EcoR1, Xba1, Spe1 and Pst1 restriction enzymes). Then Owen taught the students and conducted the following experiments together in their lab; digestion, gel electrophoresis, gel purification, ligation and transformation. On 3rd August, Owen met with the team together and discussed with them the results and compare their results to the expected results.
Modelling workshop
Hong Kong UCCKE reached out to our team for guidance with regards to learning about the basic tools for modeling. As such, on the 23rd of July, our team hosted members of the high school team for a modeling workshop where python was taught as a modeling environment.
The high school team members were provided with materials prepared by our team, and were introduced to the basics of using python as a modeling environment. Through simple demonstrations conducted along the way, concepts such as: referring to elements in arrays, defining and calling functions, and utilizing basic libraries such as “matplotlib” and “numpy” were introduced to provide a foundation for the high school team members to build on.
Cloning and transformation
The team could not synthesize an alpha-amylase gene through IDT because of the high synthesis complexity. They decided to clone the part from the iGEM kit plate but failed multiple times. After some discussion, our team offered to help them clone the parts BBa_K608002-BBa_K523001 together and into BBa_B0015-pSB1C3. The plasmid was constructed and verified.
The high school team had low transformation efficiency as they believe their competent cells have low competency. As we cannot deliver our competent cells to the high school while maintaining the cells at such a low temperature, we transformed their parts, amylase and lipase, in our lab.
Colony PCR showing transformation of the lipase plasmid was successful |
Lipase colonies |