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Team:Minnesota/Judging
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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. | 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. | ||
| - | [[Image:Plac FACS data.jpg|left|frame|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. | + | [[Image:Plac FACS data.jpg|left|frame|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. 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 is hence constitutive. Notice the peaks associated with either cell type share simalar numbers of events while the peaks from the different cells do not. The difference in events is an artifact of the differential GFP expression between the E. coli strains.]] |
Revision as of 16:39, 25 October 2010
| Home | Team | Project | Protocols | Notebook | Judging Criteria | Safety |
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Contents |
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.
| eutSK [http://partsregistry.org/wiki/index.php/Part:BBa_K311004 BBa K311004] |
| eutSN [http://partsregistry.org/wiki/index.php/Part:BBa_K311003 BBa K311003] |
| Plac GFP [http://partsregistry.org/wiki/index.php/Part:BBa_K311002 BBa K311002] |
| Strong TET promoter [http://partsregistry.org/wiki/index.php/Part:BBa_K311001 BBa K311001] |
| Weak TET promoter [http://partsregistry.org/wiki/index.php/Part:BBa_K311000 BBa K311000] |
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. 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 is hence constitutive. Notice the peaks associated with either cell type share simalar numbers of events while the peaks from the different cells do not. The difference in events is an artifact of the differential GFP expression between the E. coli strains.
A Information
B Information.
Charecterization of registry parts
about
Step 1 Information
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.
