Part:BBa_K5196008

THFMO Gibson Primer 4

This is the reverse primer for the second fragment of the THFMO gene sequence [1].

Usage and Biology

Background

The THFMO plasmid is a vector designed to express the THFMO gene in Pseudomonas putida. 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.

Primer Creation

To create our primers, we uploaded the sequences of our ordered fragments and template plasmid into NEBuilder [2]. Additionally, we input restriction sites based on the restriction enzymes we chose and the directionality of the sites and plasmid. Using this information, NEBuilder generated our primer sequences.

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 [3]. 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.

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), and 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.

Figure 4. Sequencing of mini-prepped plasmids indicating 1/3 successful Gibson transformations. The successful Gibson was the third plasmid, whose exact sequence matched, but isn'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:

Assembly Compatibility:

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

  Illegal BglII site found at 13
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]