Difference between revisions of "Team:SZU-China/Results"

We verified the translation of Carbonic anhydrase(CA) by SDS-PAGE protein electrophoresis and Coomassie blue staining. As you can see, the band near 35kDa was α-CA protein which only exist in the transformed bacteria but not in the WB800. We can conclude that the protein was translated successfully.

Fig.1 The intracellular expression of Carbonic anhydrase. This western blot result shows that we have successfully transformed gene CA into B.subtilis WB800. From the left lane to the right, there is marker, two α-CA proteins and the whole intracellular proteins of B.subtilis of WB800.

We determined CA activity according a method modified from Brownell et al. (1991). The CA activity was measured by the different rates of decrease in pH in the system involving 5ml CO2-saturated water, 10ml buffer and 500ul CA samples or Dl water. We calculated the activity according to the formula U= (T0 -Ts )/ T0 ,where T0and Ts represent time for pH change with blank and samples, respectively. By testing different kinds of CA each for several times and taking the average ,we got the CA activity per milliliter of bacteria solution.

Since the fact that CA can also catalyze the hydration reaction of ester,which can be used to assay the activity of CA, we conducted another measurement on CA according to a method from Verpoorte JA(1967).

From the above results, we may draw the conclusion that transferred strains range a higher level in CA activity.

To verify the gene gerAa for promoting spore germination, we divide the germination detection into two groups: control group (B. subtilis WB800) and gerAa group (B.subtilis WB800 that has gerAa gene with promotor pssPb). Theoretically, through the Germination detection, gerAa group should exhibit higher germination proportion and germination rate than control group, as gerAa is overexpressed. As shown in Fig.1, the germination rate and proportion varied between control group and gerAa group. gerAa group shows a high proportion in germination and rapid rate in germination. Therefore, gene gerAa is verified to be in good condition and can work efficiently.

Fig 6. The germination result of different groups. The line graph demonstrate the germination status in WB800 group(control group) and gerA group(B. subtilis WB800 that has been transformed with gene gerA). As the OD600 decrease, the endospores are germinating. The rate of decrease can descript the ability of germinate, which is mediated by germination gene.

We determined CA activity according a method modified from Brownell et al. (1991). The CA activity was measured by the different rates of decrease in pH in the system involving 5ml CO2-saturated water, 10ml buffer and 500ul CA samples or Dl water. We calculated the activity according to the formula U= (T0 -Ts )/ T0 ,where T0and Ts represent time for pH change with blank and samples, respectively. By testing different kinds of CA each for several times and taking the average ,we got the CA activity per milliliter of bacteria solution.

To verify the function of TupA for alkaline resistance in our modified baceria, we test the WB800 strain by the following steps:

At first, we conduct a gene transformation experiment of origin strain WB800, by transforming an extraordinary gene TupA. Then we collect the mono bacteria from the petri dish to culture. We also have a series of assays following up: extracting the plasmids, digesting the plasmids with endonuclease and running through DNA electrophoresis just to verify we have successfully transformed gene TupA into the B.subtilis WB800 (as shown in Fig.7).

Fig 7. The result of DNA electrophoresis. In the right lane places the marker. And the DNA sample extracted from the digested plasmid in a transformed WB800 is on its left.

Furthermore, we want to know if the WB800 we transformed can express gene TupA. So we have designed the way to verify:

1. We divide the bacteria into two groups: one is the control group (B.subtilis WB800), and another is the experiment group (B.subtilis WB800 that includes gene TupA). And culture these two groups with liquid medium.

2. Prepare four types of solid medium with the pH of 9,10 and 11 respectively .

3. When the concentration of the B.subtilis from two groups are nearly equal, take 5μL of sample and drop it on the plate(each plate has already been divided into 4 even zones) at the correct zone.(Also we have take 10μL of the sample to repeat). Culture the B.subtilis on the solid plate.

4. After 48hours, observe and verify the plate by dyeing with crystal violet before wash the plate with ddH₂O.

So we have come to the conclusion: gene TupA can regulate the alkaline resistance of B.subtilis.

Based on upstream experiment on genetic engineering level, our genetic modified WB800 successfully achieved the 3 functions needed for concrete self-repairing sysyem. Next, we will measure their true performance when embedded in the concrete as form of micro-capsule. See the DEMONSTRATE page for our downstream results.