Team:UESTC-China/Improve

team

IMPROVE PART

Overview

This year, we have improved the previous part BBa_J23100 by adding RBS and pelB-5D to make it can be used for extracelluar expression. The combination of promoter, RBS and signal peptide makes it convenient to use as a BioBrick and gives it the function of extracellular expression.
Vector design for the validation of expression system

For the verification of function, we decided to use the PETase as our reporter protein. Two plasmids were constructed.
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Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful.
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Validate the extracellular expression of PETase by SDS-PAGE

To validate the extracellular expression of PETase, the supernatant fractions of LB culture were separated by centrifugation (8000rpm,4℃). Then we used the absorbing column to do the protein concentration. Added 30mL supernatant into absorbing column, and got 1mL even less concentrated supernatant through refrigerated centrifugation(4℃, 4000g，30min).After SDS-PAGE analysis, we found that the supernatant of piGEM2016-001 which carrying pelB-5D showed a target band while no such a band was detected in piGEM2016-002 （FigXX).The result demonstrated that our improved part can increase the extracellular expression of the protein.
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Detect the activity of PETase

The activity of PETase was determined after expression of PETase in E.coli BL21 (DE3) for 24h.pNP (p-nitrophenol) assay is commonly used method for quantitative detection of lipase activity. The assay is based on the production of pNP which has a maximum absorption at 405 nm. For measuring the activity of PETase, we chose pNPB (p-nitrophenol butyrate) as the substrate which can be hydrolyzed to pNP by PETase. One unit (U) of PETase activity was defined as the amount of PETase that could release 1 mmol pNP from pNPB per minute (Kim et al., 2015). For quantitative assay, a standard curve of pNP ranging from 0-0.8 mM in 100 mM of phosphate buffer (pH 7.4) was measured (Figure 8).
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Reference