From 2009.igem.org
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| | == The Experiments == | | == The Experiments == |
| - | '''Gene expression'''
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| - | 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.
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| - | * pLacI:sRBS:mms6:dT (induced with IPTG)
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| - | * pLacI:sRBS:Lumazine Synthase:dT (induced with IPTG)
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| - | * pStrong:Riboswitch:cheZ:dT (induced with Theophylline)
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| - | * pStrong:Riboswitch:GFP:dT (induced with Theophylline)
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| - | * pBAD:RBS:TetR:dT:pTetR:mRBS:N-EYFP:dT (repressed with Arabinose)
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| - | * pBAD:RBS:TetR:dT:pTetR:mRBS:C-EYFP:dT (repressed with Arabinose)
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| - | * pBAD:RBS:TetR:dT:pTetR:mRBS:N-ECFP:dT (repressed with Arabinose)
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| - | * pBAD:RBS:TetR:dT:pTetR:mRBS:C-ECFP:dT (repressed with Arabinose)
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| - | 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.
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| - | ===Motility Assay===
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| - | The pStrong:Riboswitch:cheZ:dT construct is designed as a regulated motility construct. This gene has been designed to introduce regulated motility, to our motility knockout strain of E. coli, RP1616. In order to determine the mobility of our bacteria, we will qualitatively measure the apparent mobility of our bug in the absence and the presence of different concentrations of theophylline. We intend to do this in several different ways.
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| - | The first of which is simply a motility test. We will stab some of our bacteria into a motility media, in order to determine wether or not our bacteria are motile. A control of regular DH5a cells will be used as well.
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| - | The second is to plate the RP1616 cells with the motility construct on Plates containing different concentrations of theophylline. The cells which are plated where no theophylline is present are not expected to migrate from their original placement; Cells plated on relatively low concentrations (0.1 µM theophylline) are expected to migrate only slightly; and cells plated at moderate concentrations of theophylline (1 µM) are expected to migrate a fair distance; cells which are plated at concentration above the threshold for survival (1.5 – 2.0 µM) are not expected to survive.
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| - | The third method for determining relative motility is to view the movement of the cells via a microscope. Control cells (DH5a) are expected to have superb motility; RP1616 cells transformed with the motility construct are expected to behave differently at different concentrations of theophylline. Cells where no theophylline is present are expected to experience on a tumbling-like motion. Cells where low concentrations of theophylline are present are expected to mostly tumble, but some running is expected. Cells which are present under moderate concentrations of theophylline are expected to exhibit mostly a running motion. Cells which are present at theophylline concentrations above the threshold for survival are not expected to exhibit any motility whatsoever.
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Revision as of 16:44, 21 October 2009
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).
This essentially segregates and regulates these processes. However, bacteria lack distinct organelles, and as such, many metabolic processes intermingle. Engineering an artificial organelle, capable of containing metabolic proteins, in bacteria would thus represent a fundamental advance in biotechnology. Towards this end, the University of Lethbridge iGEM team is working towards producing microcompartments from the lumazine synthase gene, which forms 60 subunit icosahedral capsids (Seebeck et al., 2006).
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
Results
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