Difference between revisions of "Part:BBa K3699010"
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In order to explore the properties of purified mlrA enzyme, we designed BBa_K3699009 & BBa_K3699010.</p> | In order to explore the properties of purified mlrA enzyme, we designed BBa_K3699009 & BBa_K3699010.</p> | ||
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Revision as of 00:43, 28 October 2020
mlrA with 6xHis-MBP Tag
Parts for purification of mlrA--BBa_K3699009 & BBa_K3699010
In Contribution, we replaced the original promoter J23110 with the stronger constitutive promoter J23119, and new data from experiments were added to the existing part BBa_K1378001.
In order to explore the properties of purified mlrA enzyme, we designed BBa_K3699009 & BBa_K3699010.
Usage and Biology
Design
We used the T7 promoter plus 6 x His tag and MBP tag to improve the efficacy and efficiency of expression and purification. Sequences encoding fusion proteins 6xHis-mlrA (BBa_K3699009) and 6xHis-MBP-mlrA (BBa_K3699010) are improved parts of BBa_K1378001.
Maltose Binding Protein (MBP) is a member of the maltose/maltodextrin system of E.coli， which is accountable for the uptake and efficient catabolism of maltodextrins. MBP elevates the yield of its fusion partner in many cases and is often able to promote the solubility of polypeptides to which it is fused.
S-tag sequences are novel fusion peptide tags for recombinant proteins that allow detection by rapid, sensitive homogeneous assays or by colorimetric detection in Western blotting. S-tag can be used to purify target protein
Figure 1. Skeleton map of pET-mlrA and pET-MBP-mlrA. We added 6xHis tag and s-tag at both ends of the gene, and linked MBP with mlrA (BBa_K1378001). MBP is a solubilization tag, which can increase the solubility of protein in water. S-tag can be used to purify target protein.
Construction
1. Replicating the mlrA DNA through PCR, Gel Electrophoresis and Extraction of MlrA
2. The pET-DuetI and mlrA genes were digested by BamHI-XhoI double enzyme to construct the plasmid pET-mlrA.
3. Transforming the plasmid to E. coli JM109 to express the targeted protein.
4. Purifying the protein MlrA and utilizing HPLC to determine the effectiveness of the protein MlrA.
Figure 2. Plasmid Profile. pET-mlrA.
Figure 3. Plasmid Profile. MBP tag and mlrA gene are inserted into pET-MBP-mlrA.
Testing
We tried to verify whether the two plasmids were successfully constructed. It demonstrated the success of plasmid construction.
Figure 4. DNA gel electrophoresis for pET-mlrA digested with PstI-XhoI (5299bp + 888bp).
Figure 5. SDS page of JM109 harboring plasmid pET-mlrA. Inclusion bodies are formed.
Figure 6. Results of PCR and DNA gel electrophoresis. Left: PCR; Right: DNA gel electrophoresis (4222 bp, 3096 bp)
Result
The 6xHis-mlrA of pET skeleton was introduced into E.coli BL21 (DE3), and the protein containing the tagged target gene was also expressed. Although, the purification of the protein is still in process due to limited time.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3741
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 474
Illegal BglII site found at 2483
Illegal BamHI site found at 1299 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1412
Illegal NgoMIV site found at 3039 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 172