Difference between revisions of "Part:BBa K5258005"

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−	<img src="https://static.igem.wiki/teams/5258/bba-k5258005/2.jpg" width="70%" alt="Figure 2: (A) Gel electrophoresis of target gene fragment 46# and vector pET28a. (B) Gene sequencing of 46#SBD.">	+	<img src="https://static.igem.wiki/teams/5258/bba-k5258005/2.jpg" width="60%" alt="Figure 2: (A) Gel electrophoresis of target gene fragment 46# and vector pET28a. (B) Gene sequencing of 46#SBD.">
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	<caption>Figure 2: (A) Gel electrophoresis of target gene fragment 46# and vector pET28a. (B) Gene sequencing of 46#SBD.</caption>		<caption>Figure 2: (A) Gel electrophoresis of target gene fragment 46# and vector pET28a. (B) Gene sequencing of 46#SBD.</caption>

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Latest revision as of 05:17, 29 September 2024

pET28a-46#SBD

we are building and verifying the recognition ability of pET-28a+46#SBD. The general process is almost the same as the foregoing ones. Firstly, insert DNA, 46#SBD, and backbone, pET28a, are extracted from plasmids by restriction endonuclease, Xho1, and Nde1. Then, we use T4 DNA ligase to connect sticky ends on the vector and the 46#SBD. After constructing the recombinant plasmid, we transform the plasmid into the E.coli DH5α, a competent cell, by heat shock. The bacteria is cultivated, and a single colony is picked. The plasmid is extracted to verify the correctness of previous construction and has been inserted by gel electrophoresis and gene sequencing. Then, IPTG is used to induce the protein expression. SDS-PAGE is done to assess protein purity. Finally, the purified extracted target SBD proteins are tested for their binding ability with PT-DNA by carrying out EMSA.

Sequence and Features

Construction Design

First, the insert DNA (46#SBD) and the backbone (pET28a) are extracted from plasmids using the restriction endonucleases Xho1 and Nde1. T4 DNA ligase is then used to connect the sticky ends of the vector and 46#SBD. After constructing the recombinant plasmid, we transform it into E. coli DH5α (a competent cell) via heat shock. The bacteria are cultured, and a single colony is picked. The plasmid is then extracted and verified for correct construction using gel electrophoresis and gene sequencing.

Next, IPTG is used to induce protein expression, followed by SDS-PAGE to assess protein purity. Finally, the purified SBD proteins are tested for their binding ability to PT-DNA using EMSA.

Engineering Principle

pET28a-46#SBD is a new plasmid constructed using the pET-28a vector (BBa_K3521004) and a gene fragment 46#SBD (BBa_K5258002). Tuberculosis is a severe global public health crisis, with several genes in Mycobacterium tuberculosis linked to drug resistance. Detecting relevant SNPs can help doctors select effective treatments and reduce drug resistance. SBD proteins can bind and detect target DNA. In our experiment, we constructed a plasmid (pET28a-46#SBD) capable of producing the SBD protein and tested its ability to distinguish between mismatched and target DNA. We used the restriction enzymes NdeI and XhoI to isolate the pET-28a vector and the 46#SBD gene. The 46#SBD fragment was then ligated into the pET-28a vector backbone using T4 DNA ligase. Finally, the recombinant plasmid was transformed into E. coli for screening.

Cultivation, Purification and SDS-PAGE

The process of constructing the recombinant plasmid can be divided into two key parts: retrieval of the target gene and constructing the recombinant plasmid.

First, we extracted the target plasmid DNA from engineered bacteria using centrifugation and elution techniques to isolate the desired gene segment. To obtain the 46# gene, we added Nde1 and Xho1 solutions to the DNA extract. These restriction endonucleases cleaved the plasmids into two parts. The same restriction enzymes were used to isolate the pET28a backbone. Gel electrophoresis was performed to confirm that the restriction enzymes had functioned correctly, successfully separating the plasmids as expected. As shown in Figure 2, the correct target plasmid segments were obtained. The exact size of the gene is 543 bp, matching the gel electrophoresis results and confirming that we accurately extracted the target gene fragment.

Next, we excised the agarose gel slice containing the DNA fragment of interest. The target DNA fragment was obtained by heating, washing, eluting, and centrifugation. With the target DNA in hand, we constructed the recombinant plasmid. T4 DNA ligase was used to join the sticky ends of 46#SBD and the pET28a vector. The recombinant plasmid was then transformed into E. coli competent cells through heat shock. The bacteria were incubated overnight in a shaker. A single colony was selected, and the plasmid was extracted for further verification. Both gel electrophoresis and gene sequencing were performed. The results are shown below.

Characterisation/Measurement

We first extracted and purified the desired protein to test the binding ability of the SBD protein.

Protein extraction was achieved through cell lysis, starting with centrifugation of the bacterial solution at 4200 rpm for 10 minutes. Each tube was then subjected to ultrasonication at 40% power for 10 minutes, followed by centrifugation at 12,000 rpm at 4°C for 40 minutes. The supernatant was transferred to an empty centrifuge tube, and the pellet was resuspended in PBS.

The nickel affinity chromatography column was equilibrated with a binding buffer, and the clarified sample was slowly loaded onto the column. The column was washed twice: once with a low concentration of imidazole and once with a high concentration.

Next, the sample was passed through an anion exchange column, and the flow-through was collected. The column was equilibrated with 4 volumes of low-salt buffer (20 mM Tris-Cl, 300 mM NaCl). The crude protein extract was manually loaded into the column using a syringe. The flow-through was collected as Fraction A. Afterward, 1 column volume of low-salt buffer was passed through the column, and the flow-through was collected as Fraction B. Fractions A and B were combined to obtain the protein sample. The column was washed with 4 volumes of high-salt buffer (20 mM Tris-Cl, 1 M NaCl) and 4 volumes of ultrapure water, then stored in ultrapure water. The protein sample was concentrated to 2.5 mL using an ultrafiltration tube.

To remove ions from the sample, the solution was passed through a desalting column equilibrated with 4 volumes of low-salt buffer (20 mM Tris-Cl, 300 mM NaCl). The protein sample was eluted with 3.5 mL of low-salt buffer, and the eluent was retained and transferred into a clean ultrafiltration tube for protein concentration.

Finally, SDS-PAGE was conducted to assess protein purity. Samples included the supernatant, sediment, flow-through liquid, and eluent containing SBD46# (from left to right). A significant band at 20 kDa in the eluent confirmed that the protein was successfully expressed.

EMSA Test

Next, we annealed the single-stranded DNA. First, two single-stranded DNAs were added to a NaCl solution (100 mM) in a centrifuge tube. The tube was then placed in a PCR machine, with the temperature decreasing by 0.5°C every 5 minutes, starting from 95°C and lowering to 30°C. The resulting double-stranded DNA was quantified using a Nanodrop.

We prepared a 12% TAE-PAGE solution for the running buffer containing 2 mL of 30% Acr-Bis solution, 55 μL of 10% APS, 2 μL of TEMED, and ddH2O to make the total volume 5 mL. The system added 30 pmol of protein and 6 pmol of DNA at a 6:1 ratio, along with 2 mM Tris-Cl, 50 mM NaCl, 1 mM EDTA, and 10% glycerol. The mixture was incubated on ice for 15 minutes.

Vertical electrophoresis was performed in an ice bath at 15 mA/gel for 40 minutes. The gel was stained with SYBR Gold for 5 minutes and then observed under UV light. Differences between matched and mismatched DNA were evident. The matched samples displayed much clearer PT-ssDNA and PT-dsDNA bands than the mismatched samples, particularly in the rightmost lane, indicating that SBD 46# could distinguish between matched and mismatched DNA.

Future Plans

We plan to modify the sulfur modification site further, increase the 46#SBD protein concentration, and extend the incubation time. Additionally, we will investigate whether 46#SBD protein can be used as a raw material for SNP detection.