Team:Minnesota/Judging

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: it has the RNA polymerase binding region, but lacks the repressor protein binding site (Schmidt-Dannert, 2000). The modified lac promoter has strong constitutive activity, and is a good candidate for consideration by other Registry users. To make our modified lac promoter more attractive (and useful) to other teams, we have characterized it using the flow cytometry technique known as Fluorescence Assisted Cell Sorting (FACS). To assay our promoter, we used 2 different E.coli strains - JM109 and DH5 alpha-pro- the latter constitutively expresses the Lac repressor protein (LacI). FACS was used to assay promoter activity in these 2 E. coli strains under varied levels of Isopropyl β-D-1-thiogalactopyranoside (IPTG), a compound known to inactivate the LacI protein. Our rationale was that as our modified lac promoter is constitutively active, neither the presence of LacI nor addition of IPTG should have any effect on the promoter output (GFP expression). The FACS data, presented in Figure 1, demonstrates that activity of the mutant lac promoter is not affected by LacI or varying levels of IPTG, and our promoter is indeed constitutively active.

Our modified lac promoter, along with downstream EGFP, has been cloned into the submission vector pSB1C3 and submitted to the Registry of Standard Biological Parts. Before submission, DNA sequencing was performed to confirm the sequence of the mutated lac promoter.

Figure 1. Results of Fluorescence assisted cell sorting flow cytometry of E. coli transformants expressing EGFP. The fluorescence is on the horizontal axis while the percentage of events or percentage of cells counted 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 IPTG, repectively. The orange and cyan 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 similar peaks because this strain does not express LacI which is the Lac Operon repressor protein that is inactivated by IPTG. Conversely, DH5α pro cells expressed the wild-type LacI. The conformity among the peaks for the wild-type cells suggest that LacI does not bind to the promoter sequence for GFP and it is hence constitutive. Notice the peaks associated with either cell type share similar numbers of events while the peaks from the different cells do not. The difference in events is likely due to the differential GFP expression between the two E. coli strains.

Figure 2. pSB1C3 Plac GFP

Charecterization of registry parts

References

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