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Day 1 8th July

From nine o’clock to eleven and half in the morning, we’ve known our laboratory and devices around us, and most jobs have been done to prepare equipment and materials needed in the upcoming day, such as culture medium, which provides the optimum environment for bacteria to reproduce.

Day 2 9th July

From nine o’clock to eleven in the morning, we carried out Plasmid Extraction, and our purpose was to acquire DNA solution. From one o’clock to four o’clock in the afternoon, we did PCR (Polymerase Chain Reaction) for catalase’s genes by using the device-PCR Amplifier. After PCR, we finally executed Gel-electrophoresis, the main goal for this experiment is to obtain useful DNA from the large agarose pieces with several impure substances. However, the result was not satisfied enough owing to its dim light around the area of 300bp on agarose gel.

Day 3 10th July

Around 9:00am to 11:00am in the morning, we redid the PCR test due to the high temperature set previously yesterday in the process of annealing, the value of annealing temperature was way too high compared to its Tm value. Follow up, we redid process of gel-electrophoresis, to obtain a specific area of DNA on the large agarose gel. Then we moved the large agarose gel into the gel-image system, placed it inside, watched the image displayed on the screen, in the row of 300bp, quantity of genes was way too small due to its extremely dark area shown on the screen, the failed result indicated we had to redo the whole process once again. In our third try, we’ve shortened the primer in order to be more specific, and the rest of the experiment remained constant. Finally, as we placed the agarose gel inside the gel-image system, the area around 300bp was impressively bright under UV light.

Day 4 11th July

During the earlier time in the morning, we tested the concentration of DNA inside the pieces of agarose gel by using a machine called microspectrophotometer (MSP), follow up, we did digestion by using BamH1 and EcoR1, this process allowed the DNA connected to the plasmid. In addition, we’ve run the agarose gel either, to obtain target genes and carriers, this process had been done by the gel-image system. Finally, we recycled the gel, and tested its concentration by using NanoDrop microvolume spectrophotometers.

Day 5 12th July

In the morning, we’ve made the culture medium by using LB-Broth, agar, and pure water, then sterilized the solid culture medium by autoclave about one hour. After that, poured the liquid culture medium into the petri dishes, sealed petri dishes by parafilm, this whole process was carried out inside the bechtop, in order to prevent bacterial invasion. Lately, we’ve fortunatly interviewed Dr.Tan in the laboratory, we’ve asked several questions about our experiments, products, plans, digital modeling, etc. It spent almost one hour to answer all the questions we’d had, the core principle he’d told was to be patient and earnest during experiments.

Day 6 15th July

Our first mission, which is connecting carrier and DNA by using ligase, to stabilize DNA while injecting into the super competent cells, which are TOP10 competent cells. Our second mission is to complete a PPT for our exhibition in later weeks, introduction of our team, introduction of our topic, and explanation of RNAi have to be included, we spent three hours to understand the full process of RNAi, owing to its complexity.

Day 7 16th July

Firstly, the cells we’ve cultured inside petri-dish previously have fully grown a lot. And meanwhile, we have to extract one cell each time from the petri dish and inoculate it into each one of the two-mililiters centrifuge. After this process, we placed the centrifuges into the constant temperature shaker, waiting till afternoon. Secondly, we would have to do PCR, to check if the DNA was connected properly.

Day 8 17th July

Continued the procedure we’ve done previously, the following step was extracting plasmid from the TOP10 Competent cells, this process requires centrifugation. After that, we tested its concentration, and the results have shown that its concentration was high enough. Later, we’ve done digestion in our carrier, to acquire the blank carrier and the gene segments, next we sent the gene segments to the sequencing company to check the sequence of the gene segments.

Day 9 18th July

Our first mission, which was doing PCR test to amplify our genes, the RNA sample we’ve sent previously, its result has been given out. The complementary sequence matched to the original sequence. Owing to the definite result, we picked a bacterium, which is HT115, from the petri dish into a small conical flask with culture medium inside. Additionally, we dropped some antibiotics into the solution either, to make sure plasmid is copied as binary fission.

Day 10 19th July

The small conical flask has been placed inside the constant temperature shaker all night, and now we shall transfer 2ml of bacteria solution form the small conical flask into a bigger one. Providing more area for bacteria to reproduce themselves. Next up, we did the inoculation of Rhizoctonia solani, the fungi which cause corn’s sheath blight. We cut down a small fragment of solid culture base with fungi inside the petri dish cultured previously into a fresh new petri dish.

Day 11 22nd July

First thing first, we extracted dsRNA from the bacteria, HT115. There were two methods to do though. One was RNA-easy, the other one was 75%CONCEN-alcohol-sedimental method. Originally we had four methods in total, the other two, which are Trizol-reagent and phenol-chloroform method, according to ‘‘XinjiangAgriculturalSciences2021，58(4):70-71’’, both of these require toxins, therefore we abandoned them. We finally checked out the concentration of dsRNA extracted by utilizing two distinct methods, the RNA-easy method was a little bit higher than the latter. But the alcohol method was much more cheaper.

Day 12 23rd July

Unfortunately, we had to repeat the transformation process today, due to the inattention of keeping the bacteria solution in small conical flask alive previously. Therefore, we retransferred the plamid into HT115 Competent cells again, the rest of the process remained same. Follow up, we prepared two maize leave samples, we smeared our dsRNA on one of them, the another one we smeared pure water, got ready for the experiment a day after.

Day 13 24th July

Firstly, we fetched the HT115 Competent cells cultured in the constant temperature shaker last night, mixed the bacterial solution with glycerin, stocked the tube inside the -20 degrees celsius freezer. This method helps storing bacteria in long term, without losing their liveness. Our cultured rhizoctonia solani has fully grown inside the petri dish, we took most of fungi out, smeared on each of maize sample prepared last day. Observing changes of maize leaves, record distinctions between the sample with dsRNA and the sample with pure water.

Day 14 25th July

Unfortunately, both of them died as we stepped in the laboratory by left foot. Instead, we should have done another experiment, which was testing dsRNA concentration within different IPTG quantities. The result shown that 0.5mmol/L of IPTG owns the most dsRNA concentration.

Day 15 26th July

Unfortunately, both of them died as we stepped in the laboratory by left foot. Instead, we should have done another experiment, which was testing dsRNA concentration within different IPTG quantities. The result shown that 0.5mmol/L of IPTG owns the most dsRNA concentration.

Day 16 29th July

We made some liquid PDA culture medium at the first stage of the experiment, follow up we sterilized the liquid culture medium and the petri dishes. At the second stage, we poured the culture base into the petri dishes, this process was done inside the super clean work table. Finally came to the last step, we inoculated one small Rs fragment into each of the cultured petri dish. One petri dish was sprayed by pure water, the another was sprayed by dsRNA. Compared two petri dishes in the upcoming week.

Day 17 30th July

Firstly, we extracted dsRNA from constructed engineered bacteria, we carried out ethanol-precipitation due to its low price. Then we tested its concentration, 1072.16 ng/μL. We prepared Tris-HCl about 200mL, pH at 3.8, 10mM. Next experiment, which was DAB staining, this experiment would test if the hydrogen peroxide Is existed in leaves, if yes, DAB reacts with hydrogen peroxide to form brown-like stains. Last experiment, we redid the dsRNA & pure-water contrast, due to the exterior bacteria invasion.

Day 18 31st July

We made some DAB stained solution, fetched 50ml Tris-HCl made previously into the conical flask. Dissolved 0.085g of DAB stained powder in the solution, covered the conical flask by tinfoil, in order to avoid light illumination. Neutralise the solution until the powder is absolutely dissolved. Put one tobacco leaves into each two petri dishes, pour 25ml stained solution into each petri dish, used the tinfoil to cover the petri dishes, put the petri dishes into shock incubator.

Day 19 1st August

We redid the tobacco test owing to the mismeasuring of stained solution, the leaves were pierced with some holes. In addition, we redid the dsRNA & pure-water contrast due to the fail in bacteria invasion.

Day 20 2nd August

Previously, we were doing dsRNA degradation comparison, but I haven’t mentioned that before. We prepared five samples in total, each sample was prepared respectively in last Friday, this Monday, this Tuesday, this Wednesday, and this Thursday, we recollected these samples and ran on the gel, to see the quantity of dsRNA remained and the frequency of degradation. Hilariously, dsRNA was not degraded in any perspection, whatever which sample was it. Therefore, we had to do it again to make sure the accuracy of that experiment.

Day 21 5th August

Firstly，we made the Potato Dextrose Agar plate culture medium. After using the autoclave for sterilization, pour the solution into 11 petri dishes. We also inoculate the pieces of small fragments of Rs into each petri dish. One of the two dishes was covered by water and the other one was covered by dsRNA. We will check the radius of the growth range in the upcoming week. After that, we sprayed ultrapure water and dsRNA on two leaves with similar size on the same tobacco plant, and also sprayed water on the other plant with a smaller size.

Day 22 6th August

First of all, we use the spreader to scrape off the whole plate of RS and mix it with DEPC-treated water. After mix them together thoroughly, carefully smear the solution on the three leaves processed yesterday by the dsRNA and water. Then smear the solution on four leaves of rice and wait for 3 days

Day 23 7th August

We redid the first step of the DAB staining of tobacco-- dispensing the DAB solution. By adding DAB stained powder into Tris-HCl, shake it well enough and control the PH value up to 5.7. Then we divided the whole solution into two equally part, poured them into two petri dishes. After that, we need to add two leaves (one is smeared by dsRNA, the other one is smeared by water) into each of the two dishes then covered them by tinfoil and put them on the orbital shaker. We can observe the results tomorrow morning and complete the remaining steps. In the afternoon, we cultured the dsRNA together with the RS and we will measure the radius in the next two days to check whether the dsRNA effect the growth of RS.

Day 24 8th August

Following yesterday’s experiment，we went on to complete the remaining steps of the DAB staining of tobacco and check the results–the leave with dsRNA get more H2O2 compared with the one with water.