Part:BBa_K5196011
ALDH Expression Plasmid
We constructed the ALDH plasmid from a pet_28_nicA2 backbone. The backbone contains a kanamycin resistance gene and a T7 promoter prior to the ALDH sequence.
Usage and Biology
Background
The Bardwell lab provided the pET-28-NicA2 plasmid backbone containing NicA2, a nicotine metabolism gene. This plasmid includes a kanamycin resistance gene, AmpR promoter, and f1 origin of replication that is compatible with P. putida. We created a plasmid construct using this vector and a native ALDH gene from P. putida with an added Strep-tag II [1,4]. We ordered the ALDH sequence in two parts from IDT, with an added Strep-tag II to the C-terminus of the gene block to confirm protein expression via Western blot in the future [3]. The decision to divide the ALDH gene block into two fragments was necessary because IDT has strict boundaries regarding ordering fragments with high gene complexity, and therefore we were unable to order ALDH with the Strep-tag II in one piece due to its high complexity score.
pET-28-ALDH Plasmid Assembly
We performed a miniprep to isolate the donated plasmid backbone. Following this, we sequenced our plasmid samples and confirmed that we had successfully isolated the plasmid at its expected size of 7047 bases, as seen in Figure 1.
Figure 1. Sequencing by Plasmidsaurus confirmed that we had successfully isolated the correct kanamycin resistance pET-28-NicA2 plasmid. The virtual gel received from Plasmidsaurus is shown.
Gibson assembly primers were designed and ordered to fit the two fragments and vector backbone, then the fragments were amplified via PCR (Figure 2) with their respective primers.
Figure 2. PCR of ALDH fragments 1 (800 bp) and 2 (712 bp).
The PCR results were purified through PCR cleanup for further use in the Gibson assembly. To isolate the vector backbone, we digested it with the restriction enzymes XbaI and PspXI. We then performed a Gibson assembly to ligate both ALDH fragments into the plasmid vector backbone. We decided to transform our construct, as well as a positive (pET-28-NicA2 vector) and negative (digested pET-28-NicA2 vector) control, into E. coli DH5α cells through heat shock. The positive control would confirm whether our transformation protocol works, as well as whether DH5α E. coli could replicate the plasmid. The negative control would show how much background uncut plasmid existed post-digestion and thus how much would end up in our Gibson reaction mixture. After this, we inoculated separate plates with our experimental sample, positive control, and negative control on kanamycin plates. These plates can be seen in Figures 3-5.
1. E. coli DH5α + pET-28-ALDH
Figure 3. Kanamycin agar plate of successfully transformed E. coli DH5α colonies with the pET-28-ALDH plasmid. Having kanamycin resistance, these colonies were expected to grow.
2. E. coli DH5α + pET-28-NicA2 (positive control)
Figure 4. Kanamycin agar plate of successfully transformed E. coli DH5α colonies with the pET-28-NicA2 plasmid. Having kanamycin resistance, these colonies were expected to grow.
3. E. coli DH5α + digested pET-28-NicA2 (negative control)
Figure 5. Kanamycin agar plate of transformed E. coli DH5α colonies with the digested pET-28-NicA2 plasmid. Without kanamycin resistance, these colonies were not expected to grow. However, we see growth, likely due to the presence of uncut plasmid that was transformed into the competent E. coli.
The appearance of colonies on the Gibson assembly sample gave us insight into the successful Gibson. However, we see a high background on the negative plate, meaning that colonies on the Gibson plate could also contain the original pET-28-NicA2 plasmid. This means that we would need to find an effective way to screen for the Gibson plasmid.
As such, eight single-isolate colonies from the Gibson assembly plate in Figure 3 were taken to be evaluated for success. We grew mini cultures of the 8 colonies, mini-prepped the plasmid, and conducted a AflII/PspXI digestion which would create a linearized plasmid in a successful experimental pET-28-ALDH, and two bands of 5495 bp and 1552 bp in the original pET-28-NicA2 plasmid. Gel electrophoresis of the dual enzyme digestion confirmed the success of our Gibson assembly (Figure 6).
Figure 6. Gel electrophoresis of the original digested pET-28-NicA2 vector, original pET-28-NicA2 vector, and the potential pET-28-ALDH vectors from eight different colonies that have been digested (Lanes 4 and 11) or linearized (Lanes 5-10) by AflII/PspXI.
Six out of the eight sample colonies (samples 2, 3, 4, 5, 6, and 7) showed the expected bands, and of these samples, 4 and 7 were sent off to be sequenced. We can see that samples 1 and 8 seem to be the original plasmid, which is why there are two bands in lanes 4 and 11.
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
[4] Database, A. P. S. (2022, November 1). Alphafold protein structure database. https://alphafold.ebi.ac.uk/entry/Q88CR0
Sequence and Features:
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 333
Illegal PstI site found at 7558 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 333
Illegal NheI site found at 116
Illegal PstI site found at 7558 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 333
Illegal BglII site found at 222
Illegal BglII site found at 6981
Illegal BamHI site found at 413
Illegal BamHI site found at 714
Illegal BamHI site found at 1663
Illegal XhoI site found at 1416
Illegal XhoI site found at 1859
Illegal XhoI site found at 7570 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 333
Illegal PstI site found at 7558 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 333
Illegal PstI site found at 7558
Illegal NgoMIV site found at 453
Illegal NgoMIV site found at 2158
Illegal NgoMIV site found at 5201
Illegal NgoMIV site found at 5361
Illegal NgoMIV site found at 6949
Illegal NgoMIV site found at 7392
Illegal NgoMIV site found at 7501 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 7282
Illegal SapI.rc site found at 4283
Illegal SapI.rc site found at 7197
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