Team:SYSU-Software/Web-lab Validation
Wet-lab Validation
Indtroduction
In order to verify the accuracy of the genetic topology design algorithm and the practicability of the prokaryotic transcription original recommendation algorithm, we plan to use the Maloadis designer module to carry out a zero-to-zero design of gene pathways to achieve specific target functions.
Oscillations are an important type of cellular signal, characterized by periodic changes in the system over time. The process of oscillation is of great significance in cell biology. The processes of biological rhythm, movement and metabolism of cells are closely related to the process of oscillation. The basis of this cellular process is the oscillator. We started the path design with the oscillator as the target.
1.We define our objective function as the input for genenet algorithm: C = Σt(y(t)-Acos(ωt))2 where A is the amplitude and ω is the frequency of the oscillator. We got the topology structure of out genetic circuit.
2.Using the auto-fill algorithm of our prokaryotic transcription regulation parts, we got the circuit.
Fig 1:The circuit we design initially
Experiment plan
Due to the pandemic, we can't get into lab and the time left us to conduct the experiment is quiet short. What's more, we changed some materials and reagents because of some accidents. Although we haven't finish our experiments yet, we still want to carry on our wet-lab work to verify our software. We will present our results on poster, here are our experiment plan.
Fig 2: We are conducting wet-lab work.
Although our algorithm uses the whole genes length as an important criteria for parts recommendation, There are still 6 TFs in our circuit, which are too many to success. After getting advice from our advisor Tianlun Lei. We decide to conduct the circuit only containing CpxR, H-NS, MazE TF to simplify the circuit. Finally, we will construct the whole circuit to testify it.
fig 3: the simplified circuit
1.Using PCR to amplify the genes and also test the purity of gene samples.
2.Using Gibson Assembly Cloning Kit to get genes onto two Plasmid vectors (PQE-30 and PACYCDuet-1).
3.Using chemical transformation method to transform Plasmids into E.coli K12 competent cell.
4.Culturing bacteria on medium with Amp and Chl.
5.Recording the fluorescent signal strength of real time.