Team:Minnesota/Judging

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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: 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 LacI the protein. Our rationale was that as our modified lac promoter is constitutively active, neither presence of LacI nor addition of IPTG should have any effect on the promoter output (GFP expression). The FACS data, presented in Figure 1, indicates that activity of the mutant lac promoter is not affected by LacI or varying levels of IPTG, and our promoter is indeed constitutively active.

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.

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.

@@ Line 29: / Line 29: @@
 == 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). This technique was used to assay promoter activity in E. coli under varied levels of Isopropyl β-D-1-thiogalactopyranoside (IPTG), a compound known to inactivate the Lac repressor (LacI) protein. To assay our promoter, we used 2 different E.coli strains - JM109 and DH5 alpha-pro- the latter constitutively expresses LacI. Our rationale was that as our modified lac promoter is constitutively active, neither presence of LacI nor addition of IPTG should have any effect on the promoter output (GFP expression). The FACS data, presented in Figure 1, indicates that activity of the mutant lac promoter is not affected by LacI or varying levels of IPTG, and our promoter is indeed constitutively active.
+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 LacI the protein.  Our rationale was that as our modified lac promoter is constitutively active, neither presence of LacI nor addition of IPTG should have any effect on the promoter output (GFP expression). The FACS data, presented in Figure 1, indicates that activity of the mutant lac promoter is not affected by LacI or varying levels of IPTG, and our promoter is indeed constitutively active.
 {|
 |[[Image:Plac FACS data.jpg|left|frame|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.]]