Team:Heidelberg/Project dual assay plasmid

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Revision as of 23:32, 21 October 2009

Dual Assay Plasmid

Introduction

In order to control transfection efficiencies, we co-transfected pSMB_MEASURE (which expresses GFP under the control of the promoter of interest) with pSMB_REFERENCE (which expresses mcherry under the control of the JeT constitutive promoter). It would be advantageous to have both promoters and fluorescent proteins on one plasmid to achieve a 1:1 ratio of reference and measured part. This construction would allow a standardized comparison of promoter strength, due to the elimination of different transfection efficiencies. Unfortunately the repeated cloning of the construct, which is shown in Fig. 1, was not successful. Probably the plasmid was becoming too large to be assembled without advanced cloning tools.

Figure 1: The planned dual assay plasmid contained a site for promoter exchange, where one can put the promoter, that has to be tested. This promoter is upstream of a GFP gene and regulates its transcription. This gene is followed by a Jet promoter that constitutively promotes transcription of a mCherry, serving as a reference.

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 === Introduction ===
-In order to control transfection efficiencies, we co-transfected pSMB_MEASURE (which expresses GFP under the  control of the promoter of interest) with pSMB_REFERENCE (which expresses mcherry under the control of the JeT constitutive promoter).  It would be advantageous to have both promoters and fluorescent proteins on one plasmid to achieve a 1:1 ratio of reference and measured part. This construction would allow a standardized comparison of promoter strength, due to the elimination of different transfection efficiencies. Unfortunately the repeated cloning of the construct, which is shown in Fig. 1, was not successful. Probably the plasmid was becoming too large to be assembled without advances cloning tools.
+In order to control transfection efficiencies, we co-transfected pSMB_MEASURE (which expresses GFP under the  control of the promoter of interest) with pSMB_REFERENCE (which expresses mcherry under the control of the JeT constitutive promoter).  It would be advantageous to have both promoters and fluorescent proteins on one plasmid to achieve a 1:1 ratio of reference and measured part. This construction would allow a standardized comparison of promoter strength, due to the elimination of different transfection efficiencies. Unfortunately the repeated cloning of the construct, which is shown in Fig. 1, was not successful. Probably the plasmid was becoming too large to be assembled without advanced cloning tools.
 [[Image:Dual_plasmid.png|thumb|left|300px|<div style="text-align:justify;">'''Figure 1: The planned dual assay plasmid contained a site for promoter exchange, where one can put the promoter, that has to be tested. This promoter is upstream of a GFP gene and regulates its transcription. This gene is followed by a Jet promoter that constitutively promotes transcription of a mCherry, serving as a reference.</div>]]