The genetic construct for tphB was ordered from IDT. The genetic construct was assembled to be compatible with BioBrick 3A. Two vectors were used: pET24a and pSB1C3. The pSB1C3 vector contains genes coding for chromoprotein mRFP1, allowing screening for successful transformation by the color of the colony. Later, a high copy pET24a was to be used for overexpression.

Prior to incorporation, the tphB gene fragment was amplified through PCR. A gradient and touchdown PCR was performed using the gene (tphB) for DCDDH. On comparison of amplified products, the gradient PCR emerged as the amplification method of choice for tphB.

pSB1C3-mRFP1 (insert contains red chromoprotein) and pET24a-IF3 (bacterial initiation factor 3) vectors were digested separately in r-CutSmart buffer, using high fidelity PstI and EcoRI together with fast AP, to ensure de-phosphorylation of the backbone during digestion. These were then ligated with tphB (also digested with PstI and EcoRI) and transformed into chemically competent DH5α-cells. The cells were plated and grown overnight at 37ºC (see Figure 4). Note that the red chromoprotein on the cells containing the pSB1C3-mRFP1 vector, indicates religation (should our preventative measures with de-phosphorylation fail) and could thus be easily eliminated as containing our recombinant plasmid of interest. (see Figure 4).

Figure 3: Restriction digest confirming successful cloning of the construct to pSB1C3 and pET24a vectors.

After successfully transforming to DH5α bacteria, overnight cultures of colonies were prepared and plasmid DNA was subsequently harvested. This was then purified, digested, and verified as having the tphB gene in it by restriction digestion analysis (see Figure 3). Endonucleases used for restriction digestion were EcoRI and PstI. The confirmed construct was then transformed to BL21 E. coli.

Figure 4: The plates shown confirm the first successful transformation of both the recombinant pSCB1C3-tphB and pET24a-tphB to BL21 E. coli. Only bacteria possessing at least the plasmid (with the right antibiotic resistance) should be capable of growing on the respective plates (kanamycin for the pET24a-tphB and chloramphenicol for pSCB1C3). The absence of red in the colonies in the middle indicates the replacement of the chromoprotein gene in the BioBrick backbone with the DCDDHgene. Since we also treated the backbone with fast AP prior to ligation, it should help prevent religations.

Sanger sequencing by Eurofins further confirmed the successful insertion of our tphB sequence into the backbone and subsequent successful transformation and cloning experiments.

BL21 cells were grown in SOB media at 37 ºC to an OD₆₀₀ of 0.8 before addition of several varying IPTG concentrations. BL21 cells were cultured overnight with IPTG and then centrifuged. Cell pellets were re-suspended in 20 µL of 1XSDS loading buffer before loading to a 12% SDS gel.

Protein overexpression was induced according to the following conditions (see Table 1). The best overexpression conditions were those of row two in Table 1 (see below). We used a plasmid containing the gene for IF3 as a control in the case of overexpression with the pET24a vector a positive control. We chose IF3 as a control because of confirmed clear overexpression bands at 24 kDa (Forster et al. 2001) (see Figure 5).

Figure 5: SDS-PAGE results from initial overexpression efforts. Lanes underneath the heading tphB identify successful overexpression of DCDDH evidenced from thick bands.
(Reminder: tphB is the gene coding for the enzyme DCDDH)

The construct contained a polyhistidine tag for purification via affinity column chromatography at Testa Center (a cooperatively run industrial bioprocess laboratory run by Cytiva and Uppsala University) after induction of overexpression. At Testa center we had access to bioreactor in 5L scale to allow for higher cell concentrations and thereby potential for higher protein yields. The aim of working at Testa Center was to obtain a higher yield of purified TphB enzyme than that we could have obtained in the lab Confirmed overexpression clones were grown in LB medium and inoculated with IPTG for overnight growth/protein production in bioreactors generously provided by Testa Center.

After an overnight culture was grown, cells were harvested, lysed, and the supernatant collected. This was then submitted to a nickel affinity column for elution of protein via an imidazole gradient from 25mM to 500mM. No purified product was obtained due to a sharp drop in cell growth after 5 minutes within the bioreactor (see Figure 6). The scale up likely failed due to a lack of established information about optimal growth conditions for DCDDH. Since we were also working with equipment that was rather new to all of us, there is also the possibility that we set it up or ran it in such a way that resulted in the unfortunate sharp decline of our bacterial cultures There is no documented toxicity of the enzyme but the cells may have stopped growing due to internal pressures exerted on other cellular metabolic pathways by DCDDH. It may be that the enzyme began to inhibit normal processes of cellular respiration or perhaps interfered with functions of other proteins. Sadly, we did not have time for further repeats of experiments with our tphB constructs at Testa Center nor any follow-ups within our own lab due to time constraints. TphB and it’s potential purification and upscaling remains an interesting avenue for further research, given the promising overexpression reported here.

Figure 6: Graph of oxygen level (in black) for clones overexpressing DCDDH. The trajectory of the black line indicates the failure of clones to grow past 5 hours, at which time the percentage oxygen level climbs back to 100%. This indicates that the cells stopped taking in oxygen, further indicating that they discontinued to grow.