Part:BBa_K5258003

pET28a-6#SBD

Oasis iGEM built a recombinant plasmid pET28a-6#SBD. For the potential SBD protein 6#SBD, we intended to insert the gene section into the pET28a vector to form a recombinant plasmid. In the process, the T7 terminator and lacI promoter are involved as essential parts, and restriction endonucleases, Xho1 and Nde1, are used to make sticky ends. After purification of target genes and linear pET28a, T4 ligase glues the insert DNA and vector to form the new plasmid, which is later examined through electrophoresis, inserted back into the bacteria group, and delivered to the third party for further examination. After protein expression, purification, and EMSA verification, the final result will be displayed.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4402
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2622
  Illegal NgoMIV site found at 2782
  Illegal NgoMIV site found at 4370
  Illegal NgoMIV site found at 4700
  
• 1000
  COMPATIBLE WITH RFC[1000]

Construction Design

The Oasis iGEM team constructed a recombinant plasmid, pET28a-6#SBD, to express the potential SBD protein 6#SBD. We aimed to insert the gene sequence into the pET28a vector to create the recombinant plasmid. In this process, the T7 terminator and lac1 promoter were key components, and the restriction endonucleases Xho1 and Nde1 were used to generate sticky ends. After purifying the target gene and linearized pET28a, T4 ligase was used to ligate the insert DNA with the vector, forming the new plasmid. The plasmid was then verified through electrophoresis, transformed into a bacterial strain, and sent to a third party for further analysis. The final results will be presented after protein expression, purification, and EMSA verification.

Figure 1: Plasmid profiles of pET28a-6#SBD

Engineering Principle

The recombinant DNA expresses our target protein, 6#SBD. After transforming the plasmid into E. coli DH5α, the desired 6#SBD protein will be expressed. This protein is key in detecting drug resistance in Mycobacterium tuberculosis by recognizing mismatched bases.

Cultivation, Purification, and SDS-PAGE

Restriction enzymes were used to cut both the pET28a plasmid and the 6#SBD gene. With the help of ligase, the recombinant plasmid pET28a-6#SBD was formed. The new plasmid was reintroduced into bacteria via heat shock, followed by electrophoresis to confirm the success of the process.

The first step was to extract pET28a plasmids from the bacteria. Restriction enzymes Xho1 and Nde1 were used to cut the plasmids and the 6#SBD gene, creating sticky ends for the incorporation of 6#SBD. Agarose gel electrophoresis was then performed to confirm successful digestion. T4 ligase was used as a "glue" to form the recombinant DNA, pET28a-6#SBD. The 6#SBD gene fragment length was approximately 450 bp, and the pET28a vector was around 5000 bp. The exact length of the gene fragment matched the gel electrophoresis result, confirming the correct extraction of the target gene.

Figure 2: (A) Result of gel electrophoresis of target gene fragment 6# and vector pET28a. (B) Result of gene sequencing of target gene 6#SBD

After heat shock, the recombinant plasmid will be introduced into E. coli competent cells, commonly used in cloning experiments. Lysogeny broth (LB) medium is used to grow and reproduce the bacteria. After 12-16 hours, a single bacterial colony will be randomly selected for plasmid re-extraction. The selected plasmids will undergo another round of digestion, ligation, and electrophoresis to ensure success. The plasmid will then be sent to a third-party DNA sequencing institution for further analysis. The gene sequencing results match the gel electrophoresis findings, indicating that the recombinant plasmid was correctly inserted into the competent cells.

Figure 3: (A) Result of gel electrophoresis of gene extracted from the E.coli. (B) Result of bacterial colony of bacteria containing pET28a-6#SBD. (C) Result of gene sequencing of gene extracted from the E.coli.

Characterisation/Measurement

The genes on the recombinant DNA were expressed in E. coli with the aid of IPTG, producing proteins through transcription and translation. Our objective was to purify the resulting protein. The first step involved lysing the bacteria, centrifuging the sample, and collecting the supernatant filtered through a nickel affinity chromatography column. Next, the solution was passed through an anion exchange column. We equilibrated the columns using a buffer with low salt concentration (20 mM Tris-Cl, 300 mM NaCl) at a volume of four columns. The crude protein solution was injected into the anion exchange column via a syringe, and the flow-through was collected and labeled as fraction A. We then injected a low salt buffer (20 mM Tris-Cl, 1 M NaCl) into the columns and collected the flow-through, labeled as fraction B. The mixture of fractions A and B constituted the protein sample after this process. The column was then washed with a high salt buffer ddH2O, and the protein sample was concentrated at 2.5 mL using an ultrafiltration centrifuge tube. Finally, the protein sample was passed through desalting resin columns and concentrated again.

To verify successful protein expression, we conducted an SDS-PAGE test (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), a standard method to assess protein expression levels, purity, and mass. SDS and a reducing agent break bonds within the proteins, assisting in their linearization. The resulting gel image confirms that pET28a-6# was successfully expressed with high purity. The first three lanes show the supernatant, precipitate, and flow-through, containing unwanted proteins. The rightmost lane displays the eluted liquid containing our target protein, with a single band at the predicted length, indicating a successful outcome.

Figure 4: Result of SDS-PAGE

EMSA Test

The next step was primer annealing, in which the primer binds to a single template DNA strand to form double-stranded DNA (dsDNA). This step was crucial in preparing for the EMSA test. Two single-stranded DNAs were mixed in a 1:1 ratio, combined with a 100 mM sodium chloride solution, and placed in a PCR apparatus for the reaction. The resulting dsDNA product was quantified using a Nanodrop as the DNA substrate.

Next, we performed the EMSA (electrophoretic mobility shift assay). We prepared 12% TAE-PAGE using 2 mL of 30% Acr-Bis solution. Then, we mixed 30 pmol of protein with 6 pmol of the DNA substrate in a 6:1 ratio. The system also included 20 mM Tris-Cl, 30 mM NaCl, 1 mM EDTA, and 10% glycerol. The entire system was incubated on ice for 15 minutes. The precise amounts of each component were calculated for perpendicular electrophoresis, which was run in an ice bath for about 40 minutes. SYBR Gold was used to stain the DNA for 5 minutes, after which we photographed the gel under UV light.

The results showed that our protein, SBD6#, recognized the difference between matched and mismatched DNA. We then increased the 6#SBD protein concentration and extended the incubation time to detect a more distinct binding difference with mismatched DNA. Unfortunately, no significant difference was observed when 6#SBD bound to PT-DNA with different sulfur-modified sites. This may be related to the modified protein's inherent binding and mismatch recognition ability.

Figure 5: Result of EMSA

Future Plans

We plan to modify the sulfur modification site further, increase the concentration of 6#SBD protein, and extend the incubation time. Additionally, we will investigate whether 6#SBD protein can be used as a raw material for SNP (Single Nucleotide Polymorphism) detection.

References

[1] Liu, G., Fu, W., Zhang, Z., He, Y., Yu, H., Zhao, Y., Deng, Z., Wu, G., He, X. Sulfur binding domain of ScoMcrA complexed with phosphorothioated DNA PDB.

[2] Liu, G., Fu, W., Zhang, Z. et al. Structural basis for the recognition of sulfur in phosphorothioated DNA. Nat Commun 9, 4689 (2018).