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Team:Lethbridge/Project
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Project Details
Many of the metabolic processes in eukaryotic cells are localized to various subcellular compartments. For example, transcription of mRNA from DNA is localized in the nucleus; post-translational modification and folding of proteins occurs in the endoplasmic reticulum (Voet et al., 2004).
By generating a highly negative interior to the microcompartment, we hope to be able to target fluorescent proteins tagged with positively charged termini to the interior of the microcompartment (Figure 1). Utilizing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins, we will demonstrate the co-localization of these two proteins into the microcompartment (Figure 2).
For the future application of this technology we are working towards the targeting of proteins from the photosynthetic pathway into the microcompartment to optimize a biological photosynthetic fuel cell. The segregation of proteins and metabolites increases the efficiency of cellular processes and following this theme, we are working towards creating uniform nanoparticles Through the segregation of Mms6 protein within Escherichia coli (Figure 3). (Prozorov et al., 2007).
The Experiments
Gene expression The expression of a particular gene can be detected by measuring the amount of the protein product at different time points. The gene must be controlled in some sort of regulatory manner for expression to be calculable. The gene must be transformed into an expression cell strain, typically BL21 for E. coli. The cells are grown to an OD of 0.6. At this point the regulatory chemical is added to induce or repress the protein’s expression. Samples of the culture are taken at different time points. The cells are opened up and the contents are examined via some visualization technique, typically SDS-PAGE. We intend to view the expression of the following constructs in this manner.
- pLacI:sRBS:mms6:dT (induced with IPTG)
- pLacI:sRBS:Lumazine Synthase:dT (induced with IPTG)
- pStrong:Riboswitch:cheZ:dT (induced with Theophylline)
- pStrong:Riboswitch:GFP:dT (induced with Theophylline)
- pBAD:RBS:TetR:dT:pTetR:mRBS:N-EYFP:dT (repressed with Arabinose)
- pBAD:RBS:TetR:dT:pTetR:mRBS:C-EYFP:dT (repressed with Arabinose)
- pBAD:RBS:TetR:dT:pTetR:mRBS:N-ECFP:dT (repressed with Arabinose)
- pBAD:RBS:TetR:dT:pTetR:mRBS:C-ECFP:dT (repressed with Arabinose)
For the repressible constructs, as the repression of a protein is far more difficult to detect than the overexpression of a protein we intend to use fluorescence as our measurable, since in each case the protein which is repressed is a fluorescent protein. This is done in the same manner, as the expression, with samples of the culture being taken at different time points. Since we are unsure wether the intrinsic fluorescence of the cell will interfere with our measurements, we will measure the fluorescence of samples where the cells have been opened, as well as samples where they have not been opened (taken from the same culture, at the same time point). By measuring the change in fluorescence of the cells (or cell lysate) we can measure the effect of repression on the gene.
Results
