Documentation of the Development of Project
(1)PCR Polymerase Chain Reaction, PCR in short, is a biological technique that are able to produce many copies of a single piece of gene. In this case, our team’s goal is to make billions of copies of our target genes, MlrA2 and MlrA3, such that both MlrA genes would be available for later experimental processes.
(2)Gel Electrophoresis Gel Electrophoresis is a technique carried out by biological researchers for separating and visualizing DNA fragments of different lengths based on size, electrical charge, and other physical properties of nucleic acids or proteins. Under 110 volts of electricity, the negatively-charged DNA fragments are able to travel from the negatively-charged anode to the positively-charged cathode, and the lengths they have traveled are determined by the number of base pairs each type of DNA fragment contained. Hence, by comparison with the distance marked by the restriction fragments, which is the known distance MlrA2 and MlrA3 would travel, we are able to identify the specific sector of gel containing the MlrA2 and MlrA3 fragments, and discard the materials for conducting PCR. Both types of MlrA genes are collected by severing the target sectors from the gel.
Figure1. Result of Gel Electophoresis after PCR
(3)Extraction of MlrA We first add DNA binding buffer to the gel and melt the gel at 65℃. We then extract the MlrA genes from the gel with a centrifuge, a machine with a part that spins mixtures of different substances around so that substances with different masses may separate due to centrifugal force. After four rounds of revolutions, DNA then will be extracted out of the gel mixture.
(4)Concentration Testing Finally, we measure the absorbance of the resultant DNA of MlrA2 and MlrA3 with a nanodrop, the final result is 29.2nanogr am per microliter
This piece of data helps us estimate the volume of materials required in the later process of Homologous Recombination.
Molecular Cloning with DH5α Cells.
(1)Preparation of the Cultural Medium Preparation of cultural medium for the growth of DH5α competent cells, so that the MlrA genes can be successfully duplicated by the cells.
(2)Cleaving of Restriction Site Restriction endonucleases, or restriction enzymes, are enzymes that cut DNA molecules at a limited number of specific locations. As we cleave the empty plasmid with restriction enzymes, the plasmid is getting linearized such that the gene of interest, the gene containing the MlrA gene sequence, will be able to bind to the plasmid at restriction sites during homologous recombination.
(3)Gel Electrophoresis In this case, gel electrophoresis helps us identify and extract the linearized plasmid out of the plasmid-restriction enzyme mixture.
Figure 2. Gel Electrophoresis during Molecular Cloning
(4)Homologous Recombination Homologous recombination is the recombination of two fragments of DNA with similar nucleotide sequences at the corresponding fronts and ends by the homologous recombination enzyme. In this process, the homologous recombination enzyme catalyzed the attachment of the MlrA2 and MlrA3 genes to the linearized plasmids, and the formation of a complete circular plasmid.
(5)Transformation Transformation is the phenomenon defined as a change of genotype due to assimilation of external DNA by a cell. Since DH5α is a competent cell, cells with cell membranes etched with tiny holes, plasmids with MlrA genes can easily enter the DH5α cell and become a part of DH5α cell’s genotype, such that the plasmid can utilize the materials in the DH5α cell to replicate it self during culturing.
(6)Culturing Pick from monoclonal DH5α cell culture and incubate them in fluid until they have replicated enough amount of plasmids for 10 hours.
(7)Plasmid-Target Gene Testing: PCR and Electrophoresis In this case, PCR is applied to determine the presence or absence of MlrA genes on the plasmid. Due to the fact that the primer in this PCR process is paired with promoter on the gene of interest, the gene containing MlrA gene sequence, if the MlrA gene is successfully transplanted to the plasmid, the plasmids would be proliferated, and vice versa.
Conduct electrophoresis again to test whether the plasmids have been proliferated or not. As long as the distance the plasmid gene traveled matches with the appropriate gene mark, the plasmids are then proliferated and contain the MlrA genes. Since our plasmids do match with the mark, the MlrA genes are present on our plasmids.
Figure 3. Electrophoresis for Gene Testing
Line 1 to 5 contains plasmids inserted with MlrA 2 genes, Line 6 to 10 contains plasmids inserted with MlrA 3 genes.
The results showed that MlrA is inserted into the plasmids.
(8)Plasmid Extraction We then extract the plasmids from the bacterial fluid with a centrifuge after six rounds of revolutions.
(9)DNA Sequencing Decipher the sequence of the plasmids to see whether any mutations have happened to the gene. The results suggest that no mutation has happened, and the plasmids can be used for later experimental processes.
Molecular Cloning with BL21(DE3) Cells
Transfer the plasmids into BL21(DE3) competent cells so that the protein can be expressed from the MlrA genes on the plasmids by the materials in BL21(DE3).
Enzyme function test
1.Transfer E.coli from the small bottles to large flasks. Then shake the flasks until the OD (Optical Density) is between 0.6 and 0.8. The purpose of these steps is to make E.coli grow to the logarithmic phase. The cells at the logarithmic phase are good materials for metabolism and laboratory research, and also the best materials for strains, because they have the same physiological characteristics, balanced growth of the components and constant growth rate.
Figure 4. E.coli in large flasks.
2. Add IPTG to the flasks. IPTG is an extremely inducing agent widely used in the laboratory. Additionally, we also need to take a sample without adding IPTG, in order to control the variables in the following experiments.
3. Place the flasks in the shaker and induce protein expression overnight.
4. In order to test whether mlrA is expressed, we take 4 different samples. These 4 samples correspond to the supernatant solution and precipitate before and after induction, respectively. Then let these samples run the protein gel(SDS-Page gel) together for verification.
Figure 5. The result of SDS-Page gel electrophoresis
Our experimental results show that mlrA is mainly expressed in the sediment. This shows that our prediction of mlrA is a membrane protein is correct.
Purification of membrane protein mlrA
1. French press E.coli for 2 times. When this step is complete, all the cell’s membrane is broken and the cell contents are discharged. This makes it easy for us to carry out the following centrifugal.
2. 15,000g centrifuge for 10 minutes, collect the supernatant solution, and then centrifugate the solution for another hour at the force of 15,0000g. So we can make sure that the mlrA is ultimately in the precipitation after the centrifuation. The next thing we need to do is purify the mlrA from the impure protein.
3. Add buffer to the precipitation and let it re-hang, then add a certain concentration of DDM(N-Dodecyl-β-D-maltoside, this substance is able to isolate the membrane protein and maintain the normal structure of the membrane protein). Extract for 2 hours at 4 degrees Celsius.
4. The solution after extract is re-engaged for 20,000g centrifuge for 45 minutes. Because DDM is added, mlrA is present in the supernatant solution after centrifugal. Collect the supernatant solution and incubate overnight with Ni21-NTA affinity column (Qiagen) binding.
Figure 6. Ni21-NTA affinity column (Qiagen) binding
5. Purify mlrA protein with nickel column. Add the protein incubated overnight to the purification column and collect flow through. Since most of the mlrA has been combined with the binding sites on the nickel column, most of the flow through collected at this time is impure protein.
6. Gradient elute mlrA with imidazole competition agent. Imidazole will compete with mlrA on the binding sites of the nickel column. When the concentration of imidazole increases, the mlrA is gradually eluded.
Identify purification results
In order to determine whether the protein we finally purified is MlrA, we take 12 different samples from different periods of the experiment and let these samples run SDS-Page gel.
Figure 7. The result of the SDS-Page gel electrophoresis
Line 1: MlrA2 total sample
Line 2: MlrA2 supernatant sample
Line 3: MlrA2 precipitate sample
Line 4: MlrA2 precipitate sample after low speed centrifugation
Line 5: MlrA2 supernatant sample before high speed centrifugation
Line 6: MlrA2 supernatant sample after high speed centrifugation
Line 7: MlrA2 precipitate sample after membrane lysed
Line 8: MlrA2 supernatant sample after membrane lysed
Line 9: MlrA3 total sample
Line 10: MlrA3 supernatant sample
Line 11: MlrA3 precipitate sample
Line 12: MlrA3 precipitate sample after low speed centrifugation
Line 13: MlrA3 supernatant sample before high speed centrifugation
Line 14: MlrA3 supernatant sample after high speed centrifugation
Line 15: MlrA3 precipitate sample after membrane lysed
Line 16: MlrA3 supernatant sample after membrane lysed
Line 17: MlrA2 supernatant sample after membrane lysed (same as sample 8)
Line 18: Impure MlrA2 sample that first flow through the nickel column
Line 19: The sample after 5mM imidazole wash
Line 20: The sample after 10mM imidazole wash
Line 21: The sample after 20mM imidazole wash
Line 22: The sample after 200mM imidazole wash
Line 23: MlrA3 supernatant sample after membrane lysed (same as sample 16)
Line 24: Impure MlrA3 sample that first flw through the nickel column
Line 25: The sample after 5mM imidazole wash
Line 26: The sample after 10mM imidazole wash
Line 27: The sample after 20mM imidazole wash
Line 28: The sample after 200mM imidazole wash
The result of the SDS-Page gel electrophoresis is unexpected. We can only observe very small amounts of protein at the target strip of the elution samples. However, the supernatant solution and flow through do exist higher concentration of protein at the upper position than the target strip. We theoretically speculate that this higher concentration of protein may be the mlrA without the SUMO tag; but it could also be just a kind of impure protein. Because of this uncertainty, we decide to try it use both the supernatant solution and the flow through to 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.
Using HPLC to detect the effect of MlrA on the decomposition of microcystin
Figure 8. HPLC (High Performance Liquid Chromatography) HPLC (High Performance Liquid Chromatography),is a technique used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column fulled with a solid adsorbent material. Each component in the sample interacts slightly differently with the absorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column.
Figure 9: 13 samples we used to run the SDS-Page Gel
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.
We added 13 samples after 24 hours of reaction to the HPLC instrument, and the computer will generate the data and peak map separately after more than 8 hours.
To view the result of HPLC, please visit Result