After we have conceived our project, we went to the lab and tried to accomplish our expectations. Both successes and failures were taken place and were used to improve our project by iterating our practical experimental steps. On this page, we want to convince you that what exactly we managed to realize and what achievements we are able to show in our project.
Our research was essentially divided into three main sections. We will share the results we obtained from each individual step.
DNA Cloning and the Construction of Plasmid
-Successful construction of the pTOLO-EX5-mlrA plasmid
-Transformation of DH5-alpha cell and BL21(DE3)
Using the restriction enzymes Ncol and Xcol, we linearized the pTOLO-EX5 and were able to reconstruct the plasmid we want with MlrA gene we got using the homologous recombination method. Then, the homologous recombinant products were added into DH5-alpha competent E.coli for transformation.
The gel electrophoresis result of the plasmid we got
- Removal of impurities such as DNA, RNA and impure proteins by centrifugation to obtain a relatively pure plasmid
- Transformation of the plasmid into BL21(DE3) E.coli competent cells
(As you can see from the picture, we use N-Dodecyl-β-D-maltoside, which is also known as DDM, to transfer the target membrane protein mlrA from the supernatant to the membrane of DDM. Since DDM provides the membrane which mlrA depends on, with the DDM concentration increases, the concentration of the mlrA in the supernatant decreases to zero. That is the last step of protein purification.
After centrifuge the solution, to further purify the protein, we put Ni21-NTA with the supernatant and incubate overnight. The mlrA will be combined with the specific binding sites on Ni21-NTA. Then, using the method of gradient elution, while the amount of imidazole, which is a competitive agent in this step, increases, it will compete with mlrA on the binding sites of the Ni column. Therefore, the mlrA is gradually elated.
For further and detailed explain action, please view our Notebook and Experiment.
Enzyme Function Test
-Purification of the MlrA protein using nickel column
-Degradation of MC-LR (Microcystin-LR) and test the results.
To examine whether or not our purification result is correct, we ran SDS-PAGE gel to identify the results.
Surprisingly, according to the results we got, very few amounts of protein showed on the target strip of the elution samples. However, the supernatant solution and flow through did exist higher concentration of protein at the upper position than the target strip. Theoretically, we speculated that this higher concentration of protein maybe the mlrA without the SUMO tag (pTolo-EX5 is the carrier of the mlrA we designed): without the tag, it becomes lighter than usual, thus at a upper position; but it could also be just a kind of impure protein. Because of this uncertainty, we decide to let both the supernatant solution and the flow through react with microcystin to verify that if these two samples contain the MlrA we really need.
In addition to using samples we obtained in our experiments, we also used previously purified mlrA3(one specific kind of mlrA gene which is extracted from Sphingomonas sp. ACM-3962) with an initial concentration of 2 mg/ml to react with microcystin. We conducted a controlled variable experiment and tried a total of 13 reactions. In these reactions, we adjusted the MlrA concentration and the microcystin concentration respectively; the reaction temperature is 27 degrees Celsius, and the reaction time is 24 hours.
By adding the protein that binds to the Ni21-NTA to the affinity column, we were able to collect the liquid that went through. We ran the SDS-Page Gel with thirteen chosen samples to verify the results of the protein purification. It turns out, we successfully purified MlrA2&3, indiciating that it can be devoted to degrading MC-LR.
Sample 1 - The total sample of pTOLO-EX5-mlrA 2/3 in BL21(DE3) after the lysis of BL21 (DE3) cells.
Sample 2 - precipitants after cell lysis and low-speed centrifugation.
Sample 3 - supernatant after cell lysis and low-speed centrifugation.
Sample 4 - supernatant after ultra-centrifugation.
Sample 5 - The total sample after the membrane lysed.
Sample 6 - precipitants after the membrane lysed.
Sample 7 - supernatant after the membrane lysed.
Sample 8 - impure proteins that first flow through the nickel column.
Sample 9 - The sample after 5mM imidazole wash.
Sample 10 - The sample after 10mM imidazole wash.
Sample 11 - The sample after 20mM imidazole wash.
Sample 12 - The sample after 200mM imidazole elution.
Sample 13 - Blank or Buffer.
For further explanations, please visit Experiment.
High Performance Liquid Chromatography (HPLC)
To quantify whether MC-LR can be degraded from the samples, we conducted a control variable experiments,
and the following are the data we got:
The original HPLC peak map of MC-LR at the concentration of 0.5mg/ml
The HPLC peak map we get when the concentration between MlrA and MC-LR is 9:1
1.By adding the protein that we have designed, and letting it react with the MC-LR, we find that they successfully reacted just as we expected.
2.From the HPLC results, we find that as the ratio of the mlrA we have designed increases, the percentage of the reaction product increases and the amount of MC-LR decreases consequencely. This result successful indicates that the protein mlrA we have designed is able to react with MC-LR and release the product which is 160 times less toxic than MC-LR, as we mentioned in the Background.
3.Although the result of the HPLC did show that our experiment is successful basically, there are still some remained issues of the protein we have designed:
a)The efficiency of the protein we designed to degrade MC-LR still needs to be improved. We control the change in samples’ concentration by adding different volumes of the same concentration of mlrA and MCLR to the sample. As it is shown in the figure above, only when the concentration ratio of mlrA is extremely high—— for example, 9:1 or 8:2 —— can most of the MC-LR be reacted effectively. In fact, according to the data we got, the mlrA we got is able to transfer about 39% of the MC-LR to the relatively non-toxic product when the concentration ratio is 9:1. When it is applied to the real world environment, however, the efficiency needs to be boosted undoubtedly, since it is unrealistic for waterworks to reach such a high concentration ratio.
b)The future goal of our project is to invent a device to purify the MC-LR in the water using the protein mlrA we have designed. Since the protein mlrA is an enzyme, it can be reused by enzyme immobilization technology. Nevertheless, the application of the device is still not practical, due to time constraints. We hope that in the future iGEM competition, there will be teams to solve the issues we remained.
Throughout our project, we have encountered minor obstacles and failures along the way. Although these roadblocks had little impact on our end result, we did have to devote more time to recover from these unfortunate moments. One of the main failures would be the explosion of the SDS-PAGE Gel ran to identify the results of protein purification. This gel exploded in the process of heating and displayed an unclear image for examination. To solve this issue, we tried to wait for the color of the gel to dissimilate but the result was the same. We had no choice but to run the SDS-PAGE Gel again and was finally able to observe the purification result precisely.