Designed by: Taylor Remondini Group: iGEM13_Calgary (2013-09-17)
Beta-lactamase with His Tag
Beta-lactamase + His-tag
Beta-lactamase (BLA) is an enzyme frequently present in plasmids for selection. Beta-lactamase is a 29-kDa monomeric enzyme. Its enzymatic activity provides resistance to beta-lactam antibiotics such as cephamysin, carbapenems and penicillium through hydrolysis of the β-lactam ring, a structure shared by these antibiotics (Qureshi, 2007). Kinetically, beta-lactamase is simple and shows high catalytic efficiency. Also, no orthologs of BLA are known to be encoded by eukaryotic cells and no toxicity was identified making this protein very useful in studies involved eukaryotes (Qureshi, 2007). BLA has also been used to track pathogens in infected murine models (Kong et al., 2010). However, in addition to its application in eukaryotic cells, beta-lactamase efficiently cleaves a wide variety of substrates but its versatility goes beyond that; BLA preserves its activity even when fused to heterologous protein (Moore et al., 1997).
Applications of BBa_K1189009
After verifying that
) retained enzymatic activity and was able to degrade ampicillin, we performed 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).