After the team had finished the genetic design of Inland, Coastal, Marine, and Polar bacteria, time was almost run out and there were still some concerns on the performance of promoters and ribothermometers. To make sure that we could have a chance to test the uncertain parts without delaying the whole circuits’ synthesis, instructor suggested us to modularize genetic circuits and then manipulate the modules by using Gibson Assembly.
The team divided the modules into two groups. One is “promoter + operator/ RBS/Ribothermometer”. The other one is “Coding sequence + terminator”. Details are in the following form.
Actually, in Inland, Coastal, Marine and Polar bacteria, there are several overlaps in the composition of genetic circuits. For instance, Marine and Polar bacteria both contain blue chromoprotein gene; Coastal and Inland bacteria both contain yellow chromoprotein gene. Each repeating part is only shown as one module.
How can modularity help?
Firstly, the modules would be directly inserted into the plasmid pSB1C3, so that the team could hand in new parts immediately after characterizing them.
Secondly, this method ensures us to get the perfect circuits as soon as possible. It makes constructing long designed genetic circuit a more flexible process compared to the original gene synthesis and it is just the same way as an engineer welds all electronic components together to make a circuit board with expected function. Moreover, in this case, we won’t need to repeat constructing the same modules used in different circuits.
Thirdly, Gibson assembly is capable for successful assembly of multiple DNA fragments. Hence, verified modules can later be integrated with others quickly.