Part:BBa_K5196001
THFMO Expression Plasmid
This is the pJN105-THFMO plasmid. The pJN105 backbone contains gentamicin resistance and an araBAD promoter prior to the THFMO sequence.
Usage and Biology
Background
The THFMO plasmid is a vector designed to express the THFMO gene in Pseudomonas putida S16. The THFMO plasmid is versatile and suitable for transformation protocols in both Pseudomonas putida and Escherichia coli, making it an invaluable tool for genetic manipulation and biotechnological research in these bacterial species. Its design is based on extensive research and established methodologies to ensure optimal performance and reliability.
pJN105-THFMO Plasmid Assembly
The Bardwell lab provided a plasmid backbone containing NicA2, a nicotine metabolism gene, and a gentamicin resistance gene (pJN105-NicA2 Plasmid). We isolated the provided plasmid backbone through mini-prepping. Subsequent sequencing confirmed the successful isolation of the plasmid at its expected size of 7,494 bases. The plasmid has multiple restriction sites, including EcoRI-HF and XbaI, which were targeted through a restriction enzyme digest to excise NicA2. We confirmed the success of the restriction enzyme digest of our plasmid through gel electrophoresis, where we visualized two distinct bands at the expected sizes for the plasmid backbone and NicA2 (Figure 1).
Figure 1. Gel electrophoresis of pJN105-NicA2 plasmid digestion showing a ~1.5 kb band (excised NicA2 gene) and ~6 kb band (Plasmid backbone).
To construct our THFMO-encoding plasmid, we ordered the THFMO gene in three fragments from IDT (THFMO Geneblock Fragment 1, 2, and 3), as this approach minimized the risk of mutations in the gene and maximized gene block stability. These fragments were later re-ligated through the Gibson assembly to reform the fully functioning gene. We began by performing PCR and subsequent PCR cleanup on the three fragments. We visualized the resulting samples through gel electrophoresis to confirm that each fragment was the expected size (Figure 2).
Figure 2. PCR amplification of THFMO Fragments 1, 2, and 3. All three fragments ran at the expected lengths of 915 bp, 2016 bp, and 1417 bp, respectively.
We then performed Gibson assembly to ligate the fragments and the plasmid vector backbone. Before transforming into P. putida, we transformed the Gibson product plasmid into E. coli to confirm the successful cloning through the Gibson reaction and possibly troubleshoot our protocol, as E. coli has a shorter doubling time (20 minutes versus 1.8 hours). To transform into E. coli, we used heat shock transformation. We inoculated separate gentamicin plates with a negative control, positive control (for transformation), and our experimental sample.
Our negative control, consisting of our restriction enzyme digested plasmid backbone, with no Gibson assembly protocol performed, was also transformed into E. coli. Our positive control was our original donated plasmid containing the nicotine metabolism gene transformed into bacteria. We first confirmed the presence of a 10.3 kb band in mini-prepped Gibson assembly transformed bacteria, (Figure 3) then selected 3 mini-preps to sequence after seeing the expected lack of growth on the negative control plate and successful growth on both our experimental and positive control plates, allowing us to confirm the success of our Gibson assembly further (Figure 4).
Figure 3. Gel electrophoresis of mini-prepped Gibson transformed colonies. We decided to send off the plasmids in Lanes 3, 4, and 5 for sequencing.
Figure 4. Sequencing of mini-prepped plasmid indicating 2/3 successful Gibson transformations (Exact sequences matched, but aren’t shown here).
References
[1] Sales, C. M., Grostern, A., Parales, J. V., Parales, R. E., & Alvarez-Cohen, L. (2013). Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species. Applied and Environmental Microbiology, 79(24), 7702-7708. https://doi.org/10.1128/AEM.02418-13
[2] Biolabs, N. E. (n.d.). Nebuilder. NEBuilder. https://nebuilder.neb.com/#!/add/
[3] Integrated DNA Technologies. IDT. (2024, September 11). https://www.idtdna.com/page
Sequence and Features:
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 6405
Illegal PstI site found at 6777 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 6015
Illegal PstI site found at 6405
Illegal PstI site found at 6777
Illegal NotI site found at 2
Illegal NotI site found at 2915 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3715
Illegal BglII site found at 8905
Illegal BamHI site found at 5954
Illegal BamHI site found at 6182
Illegal BamHI site found at 8233
Illegal BamHI site found at 9500
Illegal XhoI site found at 4701
Illegal XhoI site found at 7107
Illegal XhoI site found at 7236
Illegal XhoI site found at 8097
Illegal XhoI site found at 8955
Illegal XhoI site found at 10274 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 6405
Illegal PstI site found at 6777 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 6405
Illegal PstI site found at 6777
Illegal NgoMIV site found at 643
Illegal NgoMIV site found at 7245
Illegal NgoMIV site found at 9642
Illegal NgoMIV site found at 9976
Illegal AgeI site found at 483
Illegal AgeI site found at 5789
Illegal AgeI site found at 6585
Illegal AgeI site found at 6928
Illegal AgeI site found at 7629 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3237
Illegal BsaI.rc site found at 6630
Illegal BsaI.rc site found at 7857
Illegal BsaI.rc site found at 9564
Illegal BsaI.rc site found at 9876
Illegal SapI site found at 5771
Illegal SapI site found at 7658
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