Part:BBa_K5258004
pET28a-25#SBD
Begin with the aim, it aims to test the ability of the pET28a-25#SBD to identify the bacteria. The process of experiments is as follows. First of all, the 25#SBD DNA chain is extracted with the restriction enzymes, Xho1 and Nde1. The restriction enzymes act as scissors, which cut the connection between the target gene and the whole DNA chain, the two ends of 25#SBD are different sticky ends, preventing self-connection and wrong-side connection. The backbone pET28a is cut using the same enzymes on two ends. So after that, the T4 DNA ligase is used to combine the pET28a and the 25# SBD, and the ends are aligned, which forms the whole plasmid ready to use. The plasmid will be sent back to the E.coli to cultivate the bacteria. When this process is done, one bacterial colony is selected for plasmid extraction. The electrolysis is then taken part to test the accuracy and whether the experiment is correct or not. The IPTG, which is a molecular biological regent, is used to express its target gene in operon. The SDS page, which is the most commonly used protein analysis technique, separates the proteins according to their molecular weight to ensure purity, the EMSA at last is used to test the ability of combination with protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4402
Illegal BglII site found at 5002 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2622
Illegal NgoMIV site found at 2782
Illegal NgoMIV site found at 4370
Illegal AgeI site found at 4502 - 1000COMPATIBLE WITH RFC[1000]
Construction Design
Through selection, three potential proteins—6#SBD, 25#SBD, and 46#SBD—were chosen for further experiments. The second cycle of experimentation focuses on 25#SBD. The objective is to test the ability of pET28a-25#SBD to identify bacteria. The experimental process is as follows:
First, the 25#SBD DNA sequence is extracted using the restriction enzymes Xho1 and Nde1. These enzymes act as molecular scissors, cutting the connection between the target gene and the rest of the DNA chain, leaving sticky ends on 25#SBD to prevent self-ligation or incorrect alignment. The pET28a backbone is similarly cut at both ends using the same enzymes.
Next, T4 DNA ligase combines pET28a with 25#SBD, aligning the ends to form the complete plasmid, which is then ready for use. The plasmid is introduced into E. coli for bacterial cultivation. Once complete, a single bacterial colony is selected for plasmid extraction.
Gel electrophoresis is performed to verify the accuracy of the experiment. IPTG, a molecular biology reagent, is used to induce the expression of the target gene in an operon. SDS-PAGE, a widely used protein analysis technique, is employed to separate proteins based on their molecular weight and ensure purity. Finally, EMSA is used to test the binding ability of the protein.
Engineering Principle
pET28a-25#SBD is composed of backbone pET28a and inserts a gene from 25#SBD. This recombinant DNA is designed to express our target protein, 25#SBD. Its production aids in detecting drug resistance in Mycobacterium tuberculosis by recognizing mismatched bases.
Cultivation, Purification and SDS-PAGE
The plasmid pET28a is first needed to serve as a carrier for the target gene from bacteria. The 25#SBD gene is isolated using the restriction enzymes Xho1 and Nde1, which cut the two ends of the target gene, leaving specific sticky ends. These same enzymes cut the pET28a plasmid, producing matching sticky ends. T4 DNA ligase is then used as a "glue" to join the pET28a backbone and 25#SBD, forming a complete recombinant plasmid.
The map of the gene encoding this SBD protein is shown in Figure 2B, with the gene length being 546 bp. We performed restriction digestion using the enzymes and successfully obtained the 25#SBD gene and the linearized pET28a backbone, as shown in Figure 2A. The gene was then subjected to gel purification to construct the recombinant plasmid.
To ensure the success of the experiments, the first test involved agarose gel electrophoresis, measuring the distances of DNA bands and making comparisons. After restriction digestion, two DNA bands remained: the linearized pET28a plasmid and the target gene, 25#SBD.
Once the recombinant plasmid was constructed, heat shock was used to introduce it into E. coli, and colonies were grown on a kanamycin-resistant plate for 12-16 hours. After plasmid extraction, further testing was performed to confirm the experiment's success. The results showed that adding kanamycin produced a single clonal colony on the LB solid plate. After selecting and culturing the single colony, the plasmid was extracted for enzyme digestion and verification. As shown in Figure 3A, the recombinant plasmid pET28a-25#SBD, when digested, produced two bands of different sizes, with the 546 bp band corresponding to the 25#SBD gene, confirming the successful construction of the plasmid.
To further verify the success of the recombinant plasmid, we sent it to a company for sequencing using the T7 universal primer. As seen in Figure 3C, the sequencing results were 100% consistent with the expected gene sequence, further confirming the plasmid was successfully constructed and ready for subsequent protein expression.
Characterisation/Measurement
The next step involved testing the binding ability of the SBD protein, beginning with its extraction and purification. Centrifugation was performed at 4200 rpm for 10 minutes to separate the protein from the solution. The tubes were then subjected to ultrasonication for 10 minutes at 40% intensity. Next, centrifugation was carried out at 12,000 rpm for 40 minutes at 4°C. After centrifugation, PBS was added to resuspend the pellet in the solution fully.
The solution was then passed through a protein purification centrifuge column. Imidazole was used for elution when the solution was poured into the nickel column, which was washed twice—once with a low concentration of imidazole and once with a high concentration. The solution was further processed through an anion exchange column to clean, purify, and separate analytes while removing contaminants such as endotoxins, host cell proteins, or ionic compounds. A low-salt buffer was used to equilibrate the solution. It was then passed through a desalting column to remove salts and low molecular weight molecules. Low-salt buffers elute the protein solution, followed by protein concentration.
Finally, SDS-PAGE was conducted to achieve high-resolution separation of complex protein mixtures. The results showed that 25#SBD, induced by IPTG, was abundantly expressed in the precipitate, with no protein detected in the supernatant. As a result, the EMSA experiment could not be performed due to the lack of soluble viable protein. In the future, we will explore methods to enhance the solubility of this protein, such as changing expression vectors or investigating protein denaturation techniques.
Conclusion
As for the SBD 25# protein, we are considering changing the expression vector or repeating the protein activation process. If successful, the EMSA experiment can then proceed.
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