From 2010.igem.org

Judging Criteria

This section will include all the work we have done to satisfy competition requirements beyond our project.

Parts Submitted to Registry

Below is out list of parts that have been submitted to the Parts Registry.

Charecterization of mutated Lac Promoter

Cloning of the ethanolamine utilization microcompartment proteins relied upon the use of a vector created in the lab of our faculty mentor Dr. Claudia Schmidt-Dannert (Johnson et al, manuscript in prep). The plasmid contains a constitutively active mutant version of the E. coli Lac promoter, which lacks the repressor protein binding sites. (Schmidt-Dannert, 2000) The flow cytometry technique fluorescence assisted cell sorting (FACS) was used to assay promoter activity in E. coli under varied levels of a compound known to inactivate the repressor protein in order to characterize the promoter as constitutive. The FACS data is presented in Figure 1.

Figure 1. Results of Fluorescence assisted cell sorting flow cytometry of E. coli transformants expressing EGFP. Number of events or cells counted is on the horizontal axis while fluorescence is on the vertical axis. Red, blue and green peaks represent florescence of GFP transformed DH5α pro cells grown at 0 mM, 0.5 mM, and 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), repectively. The orange and blue peaks represent the florescence of GFP tranformed JM109 cells grown at 0 mM and 1 mM IPTG. As expected, the peaks for the JM109 transformants had simalar peaks because this strain does not express LacI which is the Lac Operon repressor protein that is inactivated by IPTG. Notice the peaks associated with either cell type share simalar numbers of events while the . This suggests the promoter sequence used to transcribe GFP mRNA is constitutive because the cells grown with and without and that the two different cell types afford

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Charecterization of registry parts

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Step 2 Information.

References:

Schmidt-Dannert, C., D. Umeno and F. Arnold. "Molecular breeding of carotenoid biosynthetic pathways." Nature biotechnology 18.7 (2000):750-753.