<p>The genetic construct for DCDDH was ordered from IDT.
<p>The genetic construct for tphB was ordered from IDT.
The genetic construct was assembled to be compatible with BioBrick 3A.
Two vectors were used: pET24 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. A better overexpression was to be expected for the latter vector.
On successful overexpression with both pET24a and pSB1C3, no apparent difference in overexpression of DCDDH protein was obtained for either.
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 DCDDH was amplified through PCR.
A gradient and touchdown PCR was performed using the gene for DCDDH.
On comparison of amplified products, the gradient PCR emerged as the amplification method of choice for DCDDH.
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.
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pSB1C3 and pET24a were digested in r-CutSmart buffer, using high fidelity PstI and EcoR1 together with fast AP, to ensure de-phosphorylation of the backbone during digestion.
These were then ligated with DCDDH and transformed into competent DH5α-cells.
The cells were plated and grown overnight at 37ºC (see Figure 4).
Notice the red chromoprotein on the cells containing the pSB1C3 vector, confirming transformation.
Colonies were taken and grown as overnight cultures.
Plasmid prep, and extraction was performed on the O.N cultures to get the vectors out of the DH5α cells.
Vectors were thereafter transformed into BL21-cells and plated for later overexpression.
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, indicating 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).
Note that the red chromoprotein on the cells containing the pSB1C3-mRFP1 vector, indicating religation (should our preventative measures with de-phosphorylation fail) and could thus be easily eliminated as containing our recombinant plasmid of interest.
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Once the plasmid prep was complete, the validated BioBricks were offered to CaCl<sub>2</sub> competent DH5α bacteria.
After successfully transforming to DH5 bacteria, plasmid DNA was harvested.
This was then purified, digested, and verified as having the DCDDH gene in it by restriction digestion analysis (see Figure 3).
Endonucleases used for restriction digestion were EcoRI and PstI.
The ligated construct was then transformed to BL21 <em>E. coli</em>.
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 <em>E. coli</em>.
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<imgstyle="width: 50%;"src='https://static.igem.wiki/teams/4378/wiki/img/lab-enzymes/dcddh/dcddh-vectors-plates.jpg'alt="Plates with DCDDH transformed into BL21 cells + control plate">
<p><em>Figure 4: The plates shown confirm the first successful transformation of the pSCB1C3 vector to BL21 E. coli.
The BioBrick portion contains a kanamycin regulator.
The only bacteria capable of growing on plates of LB spiked with kanamycin are hence those with the construct in them.
The red colored colonies at right are a control in which the DCDDH gene has not replaced the chromoprotein gene.
The absence of red in the colonies at left indicates the replacement of the chromoprotein gene in the BioBrick backbone with the DCDDHgene.</em></p>
<imgstyle="width: 50%;"src='https://static.igem.wiki/teams/4378/wiki/img/lab-enzymes/dcddh/overexpression-tphb.jpg'alt="Plates with DCDDH transformed into BL21 cells + control plates (neg)left and (pos)right">
<p><em>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.</em></p>
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<p>Sanger sequencing at SciLife Lab and matching with established sequences (at 100% sequence match) from other authors verified DCDDH from the above experiments to be pure.</p>
<p>Sanger sequencing by Eurofins further confirmed the successful insertion of our tphB sequence into the backbone and subsequent successful transformation and cloning experiments.</p>
<p>The construct contained a tag for a polyhistidine tag for purification via an imidazole elution gradient at Testa Center after induction of overexpression.
he aim of working at Testa Center was to obtain a higher yield of purified TphB enzyme than that obtained in the lab (a shift to mg amount versus µg amount).
Overexpression clones were grown after inoculation of SOB in bioreactors generously provided by Testa Center.
<p>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.
<br><br>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.
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.
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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.</p>
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<imgstyle="width: 40%;"src='https://static.igem.wiki/teams/4378/wiki/img/lab-enzymes/dcddh/dcddh-bioreactor.png'alt="Growing status and conditions in bioreactor for DCDDH transformed BL21">
