Difference between revisions of "Part:BBa K1189031"
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===Applications of BBa_K1189031=== | ===Applications of BBa_K1189031=== | ||
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BBa_K1189031 | BBa_K1189031 | ||
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− | </a >) (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 (<a href=" https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> | + | </a >) |
+ | (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 (<a href=" https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> | ||
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189031 | BBa_K1189031 | ||
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</a >), 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.</p> | </a >), 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.</p> | ||
+ | </figure> | ||
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/parts/5/55/YYC2013_TALE_Blac_Western_Blot_White_Background.jpg"> | <img src="https://static.igem.org/mediawiki/parts/5/55/YYC2013_TALE_Blac_Western_Blot_White_Background.jpg"> | ||
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</a>) showing that we were able to express and purify our construct. | </a>) showing that we were able to express and purify our construct. | ||
</figcaption> | </figcaption> | ||
+ | </figure> | ||
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2013/thumb/3/38/YYC2013_Blac_Amp_Survival_Assay_with_protein_24h.jpg/800px-YYC2013_Blac_Amp_Survival_Assay_with_protein_24h.jpg"> | <img src="https://static.igem.org/mediawiki/2013/thumb/3/38/YYC2013_Blac_Amp_Survival_Assay_with_protein_24h.jpg/800px-YYC2013_Blac_Amp_Survival_Assay_with_protein_24h.jpg"> | ||
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</a >) were sufficient to degrade the ampicillin in the media allowing bacteria susceptible to ampicillin to grow.</a> | </a >) were sufficient to degrade the ampicillin in the media allowing bacteria susceptible to ampicillin to grow.</a> | ||
</figcaption> | </figcaption> | ||
+ | </figure> | ||
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2013/thumb/d/de/YYC2013_Blac_Amp_Survival_Assay_with_protein_3_time_points.jpg/800px-YYC2013_Blac_Amp_Survival_Assay_with_protein_3_time_points.jpg"> | <img src="https://static.igem.org/mediawiki/2013/thumb/d/de/YYC2013_Blac_Amp_Survival_Assay_with_protein_3_time_points.jpg/800px-YYC2013_Blac_Amp_Survival_Assay_with_protein_3_time_points.jpg"> | ||
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</a> 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).</p> | </a> 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).</p> | ||
+ | </figure> | ||
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/parts/5/5b/YYC2013_Blac_PenG_Assay.jpg"> | <img src="https://static.igem.org/mediawiki/parts/5/5b/YYC2013_Blac_PenG_Assay.jpg"> | ||
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</a >). 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.</a> | </a >). 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.</a> | ||
</figcaption> | </figcaption> | ||
+ | </figure> | ||
</html> | </html> | ||
Revision as of 00:32, 1 October 2013
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
Sequence and Features
Assembly Compatibility:
- 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]