View source for Part:BBa K2779912


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<partinfo>BBa_K2779912 short</partinfo>

Team UAlberta’s cloning efforts to clone our protoporphyrin IX biosynthesis pathway relied heavily on the use of visible and fluorescent markers to assemble our desired constructs. In order to characterize our enzymes, we wanted a system that would enable us to easily clone, express and purify each of our enzymes for further experimentation.

We wanted an araBAD-based expression system because: 

#It is inducible by L-arabinose and is tightly regulated by araC;
#It consists of a strong promoter  for increased expression;
#It is compatible with our preferred workhorse strain, DH10B, allowing us to perform our cloning and expression work in the same strain; and
#It is more cost-effective, since L-arabinose is more economical than IPTG.

When we initially looked through the repository, we found that while BBa_K1602055 (for Parts Page: https://parts.igem.org/Part:BBa_K1602055), which had GFP under the control of araC/pBAD, was the closest to our needs, there was no easy way for us to replace their inserted GFP with our desired genes, and there was no way for us to collect the expressed enzymes. Thus, we decided to improve BBa_K1602055.

For a full description of all the differences between this construct, which is based out of Invitrogen’s pBAD/His expression vector, and BBa_K1602055, as well as the rationale behind these modifications, please proceed to our Wiki.  Briefly,  there were three significant modifications from BBa_K1602055. The most critical modification is the inclusion of a 6xHis tag in the N-terminus. Another significant modification is the addition of a BamHI and XhoI site upstream of the GFP. Lastly, the GFP variant used in this construct contains the A206K mutation, which makes this GFP variant an obligate monomer.  

'''The translation of the 6xHis tag occurs in the +2 frame.''' This means that to ensure proper fusion of the 6xHis tag to your protein of interest, users must '''include one spacer nucleotide''' between the restriction enzyme recognition site and the first codon of the gene of interest.

'''Protein Purification Experiment'''
To demonstrate the improved functionality of this construct compared to BBa_K1602055, a protein expression and purification experiment were performed in parallel using both BBa_K1602055 and this new construct. Both plasmids were transformed in DH10B E. coli,  resuspended in LB supplemented with 0.2% L-arabinose, shaken at 37°C for at least an hour to recover, streaked out on LB-chloramphenicol plates, and incubated at 37°C overnight. 250 mL cultures supplemented with 1x ampicillin and 0.08% arabinose were then inoculated with single fluorescent colonies from each plate and grown for at least 24 hours at 37°C. 
The photos below show the cell pellets after the cultures were centrifuged down. The photo on the left shows our construct on the left and BBa_K1602055 on the right. The middle photo shows the fluorescence of our construct when the pellet is excited by blue light, while the right photo, taken under identical conditions, shows the pellet’s fluorescence under blue light.  The stronger colour and the brighter fluorescence of our construct’s pellet, when compared to those of BBa_K1602055, show improved expression. 

'''(photos go here)'''

This conclusion is further strengthened by the appearance of the supernatant following sonication to lyse the cells and release the proteins (see photos below). The left photo shows the collected supernatant of both our construct (left tube) and BBa_K1602055 (right tube).  The middle and right photos show the fluorescence of our construct and BBa_K1602055, respectively. The relative intensity of the fluorescence of the supernatants further supports the conclusion that our construct has better expression than BBa_K1602055.

'''more photos go here'''

After incubation on ice with Ni-NTA, the Ni-NTA beads were washed with PBS buffer containing 20 mM imidazole before elution by PBS buffer supplemented with 250 mM imidazole.  The two figures show the Ni-NTA beads during the washing step. While the Ni-NTA beads for BBa_K1602055 (left) show a white or slight green colour, the beads from construct (right) are indubitably green.

'''more photos'''

Furthermore, the bottom two figures show a dramatic difference in the eluted proteins, with the collected elution buffer from our construct being visibly green (left photo; right tube) and brightly fluorescent (right photo; top) compared to BBa_K1602005 (left tube in the right photo; bottom tube in the right photo). Therefore, as predicted, the His tag, as evidenced by the more pronounced green colour of the beads and the resulting eluted solution, allows for the efficient capture of the target eGFP in our construct, unlike BBa_K1602005.

'''photos'''

In this experiment, we have demonstrated that when compared to BBa_K1602005, our construct allows for increased expression and purification of proteins of interest. Though we do not demonstrate an exemplar application, we posit that this construct, by enabling the collection of the expressed GFP (or another target protein), can enable further downstream experiments and possibly facilitate quantification of upstream events in a pathway. We would also like to highlight the fact that tagging proteins of interest with fluorescent proteins like GFP facilitates expression and purification as there are visual cues that can indicate the quality of expression and purification.

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===Usage and Biology===


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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K2779912 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K2779912 parameters</partinfo>
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