iGEM Tsinghua

Parts Characterization

Overview

In order to verify our principle, we need downstream reactions. Thus, we characterized a lot of parts, catalyzing different reactions. Two examples are shown here. They all exhibit the enzymatic activity pattern as expected, similar as previous characterization.

XylE characterization

http://parts.igem.org/Part:BBa_K118021#Characterization

Before using xylE (BBa K108011) in this part for our project, we tested enzyme activity of this original part. We directly transformed the part plasmid into E. coli TOP10 strain, spread them on chloramphenicol plate, and picked single colony, which was then inoculated into 3ml chloramphenicol LB to culture overnight at 37℃.

Then the culture was separated into three parallel group and dilute to OD600 between 0.6-0.8, and different amount of glucose was added. After 1h of 220rpm shake at 37℃, the cell culture was aliquoted into 96-well plate, 100ul per well, 4 wells per group.

Varioscan Flash(TM) microplate reader was used to measure absorbance at 377nm of each well. The protocol for the instrument is listed here. The result showed significant decrease of group added 5 mM glucose than untreated group.

Rluc characterization

http://parts.igem.org/Part:BBa_K1722005

After fostering E.coli expressing Rluc at 16 degree for 10 hours, this characterization has been carried out under 37 degree, 7.5 micro moler clz, using VARIOSKAN FLASH microplate reader. To be mentioned, T7 promoter and lac operater were used here, so that 0.5 mM IPTG has been added when fostering. Three technological replicates have been shown in this plot. Clz has been added between 2 and 3 second. Finally, the light emission was normalized to OD600.