Part:BBa_K1189031
TALE-A linked to beta-lactamase with a his tag under a lacI promoter
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
Figure 1. Absorbance values at 600nm for each tube at four different time points: 0, 30, 60 and 120min. The cultures that expressed beta-lactamase (
BBa_K1189007
) showed higher absorbance levels, showing that the cells were able to grow in the presence of ampicillin.
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. Figure 2. On the left crude lysate of beta-lactamase + His (
BBa_K1189007
) from different lysis protocols: a
mechanical
and with
sucrose
, respectively. On the right, western blot of
TALE A
-linker-beta-lactamase (
BBa_K1189031
) showing that we were able to express and purify our construct.
Figure 3. Absorbance values at 600nm after 24h. Amounts from 0.1µg to 20µg of TALE A-link-Beta-lactamase (
BBa_K1189031
) were sufficient to degrade the ampicillin in the media allowing bacteria susceptible to ampicillin to grow.
Figure 4. Absorbance values at 600nm in different time points. Amounts from 1.0µg to 10µg of TALE A-link-Beta-lactamase (
BBa_K1189031
) were sufficient to degrade the ampicillin in the media allowing bacteria susceptible to ampicillin to grow.
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). Figure 5. Benzylpenicillin assay. On the top, the wells only had
TALE A
-linker-beta-lactamase (
BBa_K1189031
). Benzylpenicillin was added and after a 10-minute incubation at room temperature, we were able to observe a colour output from red to yellow (bottom row) while the control wells remained red.
Figure 6: TALE capture assay was done with TALE B (
BBa_K1189001
)and TALE A B-lac fusion (
BBa_K1189031
) and DNA (
BBa_K1189006
)
with both target sequences. If capture is successful, the B-lac is present in the well giving a colour change from pink to yellow when subjected to benzylpenicillin substrate solution within 20 minutes. The only wells that change colour are the first four wells which contain TALE B, specific DNA, and TALE A B-lac fusion and our positive control wells which are the controls for our fusion TALE A B-lac protein and our positive recombinant b-lac. All our other controls including our test using a non-specific sequence of DNA remained pink . This preliminary characterization data demonstrates that the TALEs are able to bind to DNA with specificity. Additionally it also shows that our system of capturing DNA with two detector TALEs and then subsequent reporting of the DNA’s presence works.
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 characterized this biobrick both in its ability to be a good reporter and its ability to be able to bind to DNA with specificity.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1922
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2651
Illegal AgeI site found at 677
Illegal AgeI site found at 1258 - 1000COMPATIBLE WITH RFC[1000]
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