Team:TJUSLS China/Characterization

Characterization

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Characterization


BBa_K1921021

Based on 2016 team TJUSLS_China’ s experimental results, the part we characterize this year is BBa_K1921021, which is PETase fusing with an anchor protein GCW51. We use HPLC to demonstrate this part’s function, hydrolyzing substrate BHET.

Purified PETase or induced display cells were incubated with BHET in buffer containing 50 mM glycine-NaOH (pH 9.0) for 18 h at 30 ℃ and the product was analyzed by HPLC. According to the results, we found that, compared with purified protein, the hydrolysis efficiency of surface display cells to substrate BHET was greatly improved, indicating the effectiveness and feasibility of displaying cell functions.

Figure 1


Figure 1 HPLC analysis after BHET hydrolysis by purified PETase or induced display cells. The peak appeared around 42.8 min and 44.6min are the product MHET and BHET respectively.


BBa_K1927002


AmpR, a class A beta-lactamase, is the most widely used selective marker in gene manipulation. It can hydrolyze ampicillin with specificity. Our team, TJUSLS_China, intends to screen inhibitors of the class B metallo-beta-lactamase. Due to our project requirement, we want to prove the difference hydrolysis capacity on antibiotic substrate of class A beta-lactamase(take AmpR as an example) and class B metallo-beta-lactamase(take NDM-23 as an example). So we did the contrast experiment of the two enzymes. We chose faropenem, meropenem and cefazolin as the antibiotic substrate for the two enzyme. The data is as follows.

Figure 2


Figure 2. Hydrolysis of three different antibiotics of E. coli with AmpR gene(a) and E. coli with NDM-23 gene(b). The absorption values of faropenem, meropenem and cefazolin at different nanometer wavelengths were measured at 307nm, 300nm and 273nm, respectively. The concentration of every antibiotic was 250 μM. The OD600 of E. coli with AmpR protein was 0.4, and the E. coli with NDM-23 protein was 0.02.

Figure 3


Figure 3. Hydrolysis of faropenem of E. coli with AmpR gene and E. coli with NDM-23 gene. The absorption values were measured at 307nm. The concentration of faropenem was 250 μM. The OD600 of E. coli with AmpR protein was 0.4, and the E. coli with NDM-23 protein was 0.02.

The results showed that the hydrolytic capacity of AmpR to all three antibiotics was lower than that of NDM-23, which proved that class B metallo-beta-lactamase had a broader substrate spectrum.

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