Coding
aiiA

Part:BBa_C0060:Experience

Designed by: Vinay S Mahajan, Brian Chow, Peter Carr, Voichita D. Marinescu and Alexander D. Wissner-Gross   Group: Antiquity   (2003-01-31)
Revision as of 20:42, 1 November 2017 by Orlandohramos (Talk | contribs) (→‎Applications of BBa_C0060)

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Applications of BBa_C0060

The characterization of BBa_C0060 was carried out, first, by creating a composite part (BBa_K2471000) capable of expressing the biopart of interest; this was done by the addition of a T7 promoter and RBS (BBa_K525998) and a T1 terminator (BBa_B0010) to the basic part. The design was first done in silico utilizing SnapGene®, where the program indicated that the plasmid would have a length of 3,010 base pairs. After verifying the feasibility of creating this BioBrick®, the part was synthesized using the 3A assembly method. Agarose gel (1%) electrophoresis was performed to corroborate that the obtained molecular weight coincided with the expected one.

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UNIPV-Pavia iGEM 2011

NB: unless differently specified, all tests were performed in E. coli MGZ1 in M9 supplemented medium at 37°C in low copy plasmid pSB4C5.

The AiiA enzyme activity has been characterized under the regulation of ptet promoter, assaying its enzymatic activity, with these parts:


A system of differential equations has been derived and parameters would have been identified, studying the exponential growth phase:
The four measurment parts ptet-RBSx-LuxI were quantitatively characetrized using the BBa_T9002 biosensor. Unfortunately the model parameters (kcat, kM,AiiA) were not estimated because placing the device in low copy plasmid (pSB4C5)resulted in no HSL degradation, as shown in the figures below:
A semi-quantitative information about AiiA enzyme activity is given, performing an experiment in E. coli TOP10, with the four measurement devices in pSB1A2; the results are summerized in the graphs below:
In this way a measure of AiiA activity is given, as the percentage of HSL degraded in 7 hours (measurements provided as average [stdandard deviation]):
Measurement device HSL degraded percentage
BBa_K516220 96.11 [3.66]
BBa_K516221 77.60 [2.66]
BBa_K516222 50.66 [16.87]
BBa_K516224 100 [0]

In order to evaluate the RBSs efficiency, the ratio between the percentage of the degraded HSL realtive to the measurement device with the standard RBS (BBa_B0034) was computed:
RBS RBS efficiency
BBa_B0030 0.96
BBa_B0031 0.78
BBa_B0032 0.51
BBa_B0034 1

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