TPADO: Gibson assembly endeavours
Terephthalate 1,2-dioxygenase (TPADO) was first found in Comamonas testosteroni T-2, with the activity to degrade terephthalate (TPA) into 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD).
The first study to report purification of a component of TPADO was by Schläfli et al. (1994), in which the oxygenase component was isolated and studied.
Shigematsu et al. (2003) also successfully isolated the oxygenase component containing two subunits and determined the DNA sequence of it in Delftia tsuruhatensis T7.
With the identification of the TPA degradation gene clusters, TPADO was more widely discovered and studied.
Later in other strains (C. testosteroni YZW-D (Wang et al. 1995), Rhodococcus sp. DK17 (Choi et al. 2005), Rhodococcus jostii RHA1 (Hara et al. 2007), Acinetobacterbaylyi ADP1 (Pardo et al. 2020) and Pseudomonas umsongensis GO16 (Narancic et al. 2021)),
TPADO was also identified and studied with the second component (TphA1) identified as the reductase component.
The conversion of TPA to DCD is catalysed by TPADO and its large oxygenase subunit TphA2, its small oxygenase subunit TphA3 and its reducates component TphA1, and utilises NADPH as an electron donor (Sasoh et al. 2006).
Due to the large size of TPADO subunits and TphA (in total ~3.6 kb), three shorter inserts corresponding to tphA1, tphA2 and tphA3 were designed and ordered with overhangs for Gibson assembly. The overhangs were designed to overlap the BioBrick prefix and suffix at the ends and overlap the connections between the inserts, all overlaps with a length of between 17-27 bp. A schema of the inserts (with overhangs) and the intended Gibson assembly of them is illustrated in Figure 1 below. The intended finalised product would be the inserts combined and inserted into a pET24a vector.
Figure 1: Schema over construct design for TPADO- the three inserts with overhangs and the Gibson assembly of them. The combined inserts would serve as an insert in a pET24a vector.
Three attempts of Gibson assembly, which would produce a functional plasmid containing all the TPADO genes, and one attempt to join the DNA segments through PCR reaction, were performed. The protocol used for the Gibson assembly is stated in General protocols and was done with equimolar amounts of the three inserts and pET24a vector (when included).
Attempt 1 - Gibson assembly of inserts and BioBrick ligation
The fragments containing tphA1, tphA2 and tphA3 genes came in a concentration of 63.7 ng/µL, 76.3 ng/µL and 38 ng/µL, out of which 1 µL was taken from each, for Gibson assembly, performed according to protocol.
This resulted in an estimated concentration of 7.5 ng/µL, accounting for the overhangs.
This low amount of overall tphA1A2A3 fragment would cause more issues further down the experiment, as all calculations had to be adjusted to make up for this issue.
To insert these genes into a pET24a vector, the construct resulting from the Gibson assembly and the vector were digested using EcoRI and PstI.
This would allow pET24a to be ligated to the BioBrick prefix of tphA1 and suffix of tphA3.
Due to the low concentration of the tphA1A2A3 fragment, ligation to the pET24a vector could not be assessed with gel analysis.
The resulting construct was directly transformed into competent DH5a E. coli cells, which were left to grow overnight (in LB-kanamycin).
No colonies were shown to grow after this period which indicated unsuccessful Gibson assembly.
Attempt 2 - Gibson assembly with inserts and vector
The second attempt at Gibson assembly was performed with the three inserts and pET24a vector, with reaction components (buffer, enzymes etc.) from previous iGEM Uppsala teams. The vector was prepared from a pET24a plasmid digested with EcoRI and PstI. To screen for the results, transformation of the assembly mix and positive control into DH5α cells was done. The control plates for both the Gibson assembly and transformation showed growing positive controls and no growth on negative control for transformation. However, the plates containing cells transformed with the Gibson assembly construct did not show growth which indicated unsuccessful assembly again.
Attempt 3 - Gibson assembly with inserts and vector #2
A third Gibson assembly was performed with the three inserts and pET24a vector, with newer reaction components.
The vector was prepared from a pET24a plasmid which was digested with EcoRI-HF and PstI-HF in rCutSmart buffer (all Thermo Fisher).
Digested DNA was then separated on a 1% agarose gel and extracted with GeneJET Gel Extraction Kit from Thermo Scientific, with the modifications of 11 μl of elution buffer to concentrate the DNA as much as possible, as well as ~5 min incubation at room temperature with elution buffer before the final centrifugation step.
The concentration of the digested vector (measured with Nanodrop) was very low, which led to a total volume of 30.62 µL Gibson assembly reaction mixture instead of the 20 µL in the protocol.
Screening transformation was done with NEB 5-alpha competent E. coli (high efficiency) cells (from New England BioLabs) that were transformed with 3 μl of the two samples, according to the protocol from the manufacturer of the cells.
Controls for the transformation were 5 µL dH2O (negative) and 8.78 ng pET24a (positive). 100 μL of each incubated mixture were plated on kanamycin plates (except for Gibson positive control, plated on kanamycin + ampicillin), and 50 μL and 20 μL (+30 μL SOC) of the assembly mix were plated on kanamycin plates as well.
The result showed functioning controls but only two colonies in total for the plates with the assembly mix (see Figure 2).
Figure 2: Transformation of Gibson Assembly products. From left to right; Gibson Assembly positive control, transformation positive control, 100 μL Assembly mix and 20 μL Assembly mix. The colonies on the two rightmost plates are marked with black circles. The negative control and 50 μL Assembly mix were both without colonies.
The two colonies were analysed with colony PCR. The programme and reaction mixture used were for colony PCR, with an extension time of 54 s (based on the intended 3.6 kb construct). The products from the colony PCR were analysed with an agarose gel analysis, which showed that the Gibson assembly attempt failed to produce pET24a plasmid with the TPADO genes (see Figure 3).
Figure 3: Colony PCR of Gibson assembly colonies. No bands at the expected 3.6 kb was present and indicated failed assembly.
Attempt 4 - PCR
Another attempt to connect fragments TphA1, TphA2 and TphA3 of TPADO genes was to join them using PCR, since the fragments had overlapping ends and was hypothesised to therefore act as primers for each other. Based on the annealing temperatures for the intended primers (see Table 1), a gradient PCR was done with annealing temperatures in a 63 - 71 oC gradient.
Table 1: Primer annealing temperatures of different segments. Temperatures calculated by Thermo Fisher Tm calculator.
| Segment | Tm temperature (oC) |
|---|---|
| Forward primer | 68.5 |
| Reverse primer | 67.3 |
| TphA1A2 overlap | 70.7 |
| TphA2A3 overlap | 64.3 |
The programme used was the colony PCR with 2 min extension time, combined with said annealing temperature gradient.
Reaction mixture was done as for colony PCR but template DNA consisted of 0.7 ng of each of the three fragments.
The PCR results were visualised on a 1 % agarose gel, which showed no bands around 3.6 kb and therefore that this attempt to connect TphA1A2A3 also failed.
Note that unfortunately neither negative (e.g. dH2O) or positive control (e.g. pET24a as DNA template) were used in the experiment so the success of the actual PCR programme could not be determined.
Figure 4: PCR results visualised on a 1% agarose gel. Expected fragment size from a correctly assembled construct was 3.6 kb (corresponding to combined TphA1A2A3 construct), but are not present in any sample. Note that neither positive or negative control was used in this experiment.
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