Difference between revisions of "Part:BBa K1189031:Experience"

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Applications of BBa_K1189031

For information on TALEA refer to BBa_K1189022.

This part was built to function as both our detector (TALE A) and our reporter (beta-lactamase). The part was built with the lacI IPTG inducible promoter J04500, with RBS, and it has a His-tag for protein purification.

TALE A was inspired by the award winning TALE A from the award winning 2012 Slovenian iGEM project. The iGEM Calgary 2013 used this TALE and its associated DNA binding sequence to build a proof of concept TALE based DNA detector. In the case of BBa_K1189031, the Calgary team used beta-lactamase as a reporter enzyme to indicate when the TALE is bound to DNA.

Applications of BBa_K1189031

In addition to that, we have purified our beta-lactamase ( BBa_K1189007 ) and our mobile TALE A linked to beta-lactamase construct ( BBa_K1189031 ) (Figure 2) and we have demonstrated that beta-lactamase retained its enzymatic activity for both proteins. We repeated a variation of ampicillin survival assay where we pretreated LB containing ampicillin and chloramphenicol with our purified TALE A linked to beta-lactamase ( BBa_K1189031 ). We then cultured bacteria in the treated LB that only carry resistance to chloramphenicol. Therefore, the bacteria are only able to survive if the our isolated protein retained its enzymatic abilities. We can show that the bacteria susceptible to ampicillin was able to grow in the presence of our purified construct protein ( BBa_K1189031 ), which means that we are expressing and purifying functional protein which is degrading the ampicillin (Figures 1 and 3). Figure 3 shows the OD at 24 hour time point from culturing where Figure 1 shows OD change over time. Both graphs show an increase in OD for cultures pre-treated with our protein demonstrating our protein is functional.

After verifying that TALE A -linker-beta-lactamase ( BBa_K1189031 ) retained enzymatic activity and was able to degrade ampicillin, we performed a colourimetric assay using benzylpenicillin as our substrate. We were able to see a colour change from red to yellow. This is because there is phenol red, a pH indicator, added to the substrate solution. Beta-lactamase hydrolyzes benzylpenicillin to penicillinoic acid, which changes the pH of the solution from alkaline to acidic. This pH change causes the phenol red to change from red to yellow. Our negative controls, to which benzylpenicillin was not added, remained red. We can also see the colour change correlate to the amount of purified TALE A linked to beta-lactamase present in each sample (Figure 5).

Additionally, we have demonstrated the same pH change of benzylpenicillin to penicillinoic acid by the TALE A β-lactamase fusion (BBa_K1189031) with bromothymol blue. The pH change causes the bromothymol blue to change in colour from blue to yellow as it gets more acidic (Figure 11 & 12). In the presence of TALE A β-lactamase fusion we see the colour change to yellow whereas the negatives not containing TALE A β-lactamase fusion remains blue (Figure 11). We did a kinetic analysis at 616 nm every 30 seconds and as the blue colour disappears, the absorbance at 616 nm decreases (Figure 12). Therefore, the lower the decrease the better the TALE A β-lactamase fusion reporter activity. Our kinetic activity shows that the biggest decrease is in our positive recombinant β-lactamase followed by the TALE A β-lactamase fusion at 10 micrograms. This decrease is lessened as we decrease the amount of TALE A β-lactamase fusion. We can also show that in our negatives with no TALE A β-lactamase fusion we do not have a decrease in absorbance. We have demonstrated the reporter activity both qualitatively (Figure 10 & 11) and quantitatively (Figure 12).

In order to demonstrate that we can successfully capture target DNA with two TALEs we did a capture TALE assay. TALE B was incubated with DNA containing target sites for TALE A and TALE B and blotted on nitrocellulose. After blocking and washing, TALE A β-lactamase fusion (BBa_K1189031) was added to the nitrocellulose strips. The strips were transferred into a 96 well plate to which a benzylpenicillin substrate solution with phenol red was added. If the TALE A β-lactamase fusion bound to the target site for TALE A then the solution will change colour from pink to clear. If TALE A β-lactamase fusion was not present, the solution will remain pink. We can show that the first four samples which have TALE B with DNA for TALE A and TALE B show a colour change indicating that we are successfully capturing the target DNA and reporting it. Furthermore we can also show that when we add non-specific DNA we do not see a colour change demonstrating that we can successfully capture only specific DNA and report its presence with an easy visual colourimetric output.

This assay shows that we can capture our target DNA with two detector TALEs with specificity . Additionally, we can report whether that DNA has been captured and is present in the sample, which is a very important concept for our sensor system.

To conclude, we have demonstrated that we can build, express, and purify this part. This biobrick has also been characterized to show both its ability to be a good reporter and its ability to be able to bind to DNA with specificity.

To further see if the TALE capture assay works (figure 17). In order to do this, we blotted TALEB fused to a K coil (BBa_K1189030) on a nitrocellulose strip. Subsequently the strips were blocked with 5% skimmed milk in TBST buffer. The strips were then washed and soaked in a solution containing [A] and [B] on the same plasmid(BBa_K1189006). The strips were then washed again with TALE binding buffer and soaked in a solution containing TALEA+β-lac (BBa_K1189031). Then a dot blot was done on the strips with β-lactamase primary antibody and an hrp conjugated secondary antibody. This assay demonstrated that the TALEs can capture DNA.

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