Team:Exeter/Notebook/August17
Monday, 17 August
Lab Work
Anna and Bethan used one pot cloning and the thermocycler to clone the DNA we had frozen on the 11th.
For E. coli, we had one RBS, two terminators and two promoters. For B. sub, we had one RBS, one terminator and three promoters. We had six coding sequences we needed to test out; we therefore came up with the following well plate plan to ensure that we tested each coding sequence with all possible combinations of promoter/RBS/terminator for each bacteria:
(We did not combine the T7 promoters/terminators with P_J23100 or B0015 because we assumed the T7s would only work together – our supervisors later corrected us on this).
We made 15 lots of master mix (consisting of destination vector, Fast Digest buffer, ATP, Bsal, ligase & water) for E. coli and 20 for B. sub so that we would have some spare, then added the appropriate entry vectors to each Eppendorf tube and ran the mixes through the thermocycler under the following conditions:
- 37 °C for 2 min
- 22 °C for 3 min
- Repeat above steps for 25-50 cycles
- 37 °C for 5 min
- 22 °C for 5 min
- 65 °C for 10 min
- 10 °C hold
Science Communication and Outreach
Feedback from our first questionnaire suggested that people didn't understand the biology behind our project, so we posted another infographic about synthetic biology, this time explaining which coding sequences we planned to express in our bacteria and why. We started writing infographics about the process of genetic engineering and about the individual proteins and enzymes our project uses, to clarify the scientific process further.
Collaboration with Experts/Industry
Dr Rebecca Hooper of the British Lime Association responded to our email stating that she would be happy to answer our questions and discuss our project in more detail.
Anna and Matthew had a zoom call with Dr Sam Stevens at 9:30 in the morning and spoke about X-Ray crystal diffraction and other ways that we could identify calcium carbonate and distinguish between different crystal structures.