Our general understanding of genetic engineering principles come from our textbook actually. Just by coincidence, we were learning genetic technologies during the time of designing our experiment. The teacher in the school and our instructor provided us with support during the process.

1. The choice of GOI:

After we determined to do experiment using chitinase gene. There are actually hundreds of chitinases in the world, from insects, fungi, and plants. The activity of chitinase various between the optimal pH and temperature. We pay more attention to the optimal pH value because we need to consider more about the pH condition inside the aphids’ gusts. We need to make sure the chitinase will not be digested easily and have time to decompose the chitin inside the pests. Finally, we choose the gene from plants because it has a higher probability to express successfully in plant cells.

2. The choice of plasmid:

he reason for our choice of plasmid is not difficult —— to find out a binary system vector which can be expressed in plant cells. At the same time, we considered about the budget and financial capacity. At first, we decided to use pMD-18T vector which we saw another former research used successfully. However, we didn’t find the vector successfully in biology companies, so we have to re-choose our object. After realising that not all the materials we want may be approachable on the market, we collected all the material available in each company and try to choose a new one from those. At the same time, as we already added restriction sites on our GOI, it’s better for us to choose the vector containing the same restriction sites and the same sequence of the sites as before. Then, we found pCAMBIA2301, which is only a third of the original budget and has EcoRI and Hind III in the desired sequence. Also, the distance between restriction sites is enough to eliminate the possible mal-function of enzymes;

3. The enzymes:

Apart from the necessity T4 ligase, we also need to choose the restriction enzymes. We chose the restriction enzyme based on the restriction sites on the plasmid. The two sites on the MCS should have a certain distance so that the enzymes can function well. Luckily, the plasmid contains EcoRI and HindIII, which are the two most commonly used enzymes in researches;

4. Primer design:

Just before our experiment, we found that we added the restriction sites on the GOI in opposite direction, which will lead to mal-expression of GOI when translated. After the hint of the instructor, we re-designed out primers. We now contain two pairs of primers. The first pair removes the original restriction sites at the end of the GOI and the next pair adds the new restriction sites in correct order. However, when we did the ligation, it continued to fail even after we tried to increase the concentration of GOI. Then, the seniors in the lab found that there was an error in our primers —— they didn’t contain protective base pairs. After correcting the mistake and re-synthesizing the primers, the experiment carried on.

5. The choice of E.coli:

E.coli DH5α is of the most commonly used bacteria stain in the lab. Also, pCAMBIA 2301 replicates well in this bacteria strain, which increases the efficiency of the lab design;

6. The choice of agrobacteria electroporation competent cell GV3101:

This cell strain was applied from previous research papers so it makes us easier to design the protocol in detail;