This Year team CAU_China 2020 attained and designed these basic parts from former research and ourselves.
PepperRNA basic part
Pepper RNA is a kind of RNA molecular which can bind with fluorescence and emit specific length of light. From the research, pepper RNA has been created in 1X, 2X, 4X, 8X and 16X with different length sequences.(Picture below is from fr-biotechnology website and we get their access)
In the research, pepper RNA is a technic that can dye the in vivo RNAs in the cell and trace them. Comparing with our fluorescent RNA, pepper RNA shows low background fluorescence in live cells and is monomeric, stable and bright. In our project, pepper RNA is employed to measure the concentration of dsRNA by inserted in the middle of sensing and anti-sensing sequence.
To standardize it, we plotted a standardize curve of 2Xpepper RNA. We made a series of pepper RNA solution sample and then added same volume of HBC 525nm fluorescent. After 20 minutes, we’ll examine the light intensity of the solution with spectrometer and record the data.
For team who will need measure specific RNA, they can bind pepper RNA with the target sequence. By adding HBC fluorescent, team can observe target RNA.
dsRNA basic part
In our project, we designed three basic part, targeting to three different genes of locust. In our design, we selected Locust migratoria gene trypsin-like serine protease, chitin synthase and ATPase subunitH gene as our target gene. By using specific program to design dsRNA sequence, we finally designed our three basic parts: TSP dsRNA sequence, CHS dsRNA sequence and ATPase dsRNA sequence.
We also measured their effect to locust by injection experiment.
As the result shows:
Comparing with the WT and control group (by injecting DEPC water) locust survival rate, we used LT50 to measure dsRNA effect. From the diagram above, CHS dsRNA injection has a great effect to locust which CHS experiment group LT50 is approximately 4days.
Here is the survival rate that we measure during the whole experiment process.
From the reference we searched, gene ATPase and TSP can also affect locust survival rate and interfere their gene expression which ATPase RNAi resulted in 96.7% mortality in 11days and TSP RNAi resulted in 100% mortality in 7 days. All their experiment is done at 5th stage instar locust larvae.
There are still many problems to conquer in our project Abaddon Remedy. We will do further research on proportion between yeast and Metarhizium and a dissolvent for the mix. In addition, our locust bio-pesticide works slowly comparing to common chemical pesticide.
In our light control system part, we employed CarH-VP16 protein.
This part is a composite part that we'll combine it with our plasmid PGBKT7 and transform it into Sacchromyces cerevisiae. This part is also a light control system protein and regulates the expression of dsRNA.
In our project, we’ve already transferred our CarH composite part into yeast. We’ll do further research to test whether it will work well in yeast.
This year, we attained part from team CAU_China 2019 and improved its part INP-CEN (BBa_K3279008). By using RT-PCR, we extracted AGA2 cDNA from yeast
In order to enhance yeast ability on fermentation, especially in cellulose degradation so that farmer could reuse the yeast powder to get more yeast biopesticide. We employed yeast surface performance protein AGA2. The endoglucanase fused with AGA2 protein can anchor in the yeast cell wall. We’ve already the part and inserted it into our plasmid. After we transferred AGA2-CEN part into our engineering yeast, we’ll test its endoglucanase enzyme activity. In addition, we fused a 6His-tag on the C-terminal of endoglucanase so that we can use immunofluorescence staining to identify the position of CEN protein.
Metarhizium acridum part
We obtained PMaGPD promoter from Metarhizium acridum. Comparing with GPD promoter for fungi plasmid, obtained from Aspergillus nidulans, this promoter was analyzed by 5’ -deletion strategy with GUS gene as reporter gene. The analysis from the reference reveals that PMaGPD includes the 1.7 kb region upstream of the start codon of the Magpd gene. According to the experiment, deletion from the -1691bp to -1463bp didn’t significantly affect the PMagpd activity. We obtained Pmagpd sequence by using PCR and cloning from the extract Metarhizium acridum’s genome. Here is our electrophoresis picture of PMagpd with 1463bp.
Our project aims at creating a part that yeast and Metarhizium can express dsRNA on their own and finally result in killing locust. So we design dsRNA composite part for each.
For yeast composite part, we created composite part BBa_K3467012. This part is composed of ADH1 promoter for yeast, CarO operon and ATPase dsRNA sense and antisense sequence with pepper RNA sequence in the middle.
We’ve attained this composite part and ready to insert it in our plasmid.
For Metarhizium acridum composite part, we created composite part BBa_K3467014. This part is composed of PmaGPD promoter (BBa_K3467005), ATPase dsRNA sense and antisense sequence with pepper RNA sequence in the middle and ADH1 terminator.
Here is a summary of CAU_China 2020 team part:
|BBa_K3467000||2X pepper RNA||Wang Ran||144bp|
|BBa_K3467001||TSP dsRNA sequence||Wang Ran||423bp|
|BBa_K3467003||CHS dsRNA sequence||Wang Ran||669bp|
|BBa_K3467004||ATPase dsRNA sequence||Wang Ran||914bp|
|BBa_K3467005||Promoter of Metarhizium acridum GPD||Wang Ran||1483bp|
|BBa_K3467006||CarO binding site||Wang Ran||174bp|
|BBa_K3467010||ADH1 promoter for yeast||Wang Ran||705bp|
|BBa_K3467002||T7+TSP dsRNA sequence||Wang Ran||450bp|
|BBa_K3467013||T7+CHS dsRNA sequence||Wang Ran||696bp|
|BBa_K3467003||CHS dsRNA sequence||Wang Ran||669bp|
|BBa_K3467014||Metarhizium acridum dsRNA sequence||Wang Ran||2710bp|
|BBa_K3467012||ATPase dsRNA composite part||Wang Ran||1894bp|