From 2013.igem.org
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| | content.titleLong = "Results"; | | content.titleLong = "Results"; |
| | content.summary= "Results"; | | content.summary= "Results"; |
| - | content.text="I)Experimental design – Proof of Concept</br>1.1)Reporter</br> | + | content.text=""; |
| - | For our proof of concept we needed a reporter protein with an easy Readout, which is reliable and does not interfere with our light sensitive system. Since fluorescent proteins are very easy to use and offer a variety of readouts (e.g. FACS, plate reader measurement) we decided to use one of them as a reporter. Of course we needed a fluorescent protein that would not lead to an activation of our system when excited for the read out. The light sensitive domain we are using – LOV2 from Avena sativa – is activated by a wide spectrum of light ranging till about 500nm. Therefore we searched for proteins being excited by light with a wavelength higher than 500nm. Since we wanted to have the excitation wavelength as far as possible away from the excitation wavelength of LOV we made use of the red fluorescent protein mCherry. Especially the short maturation time (~30min) made it highly favorable for our use in comparison to alternatives like dsRed since this enables a pretty fast reaction time and therefore a good temporal resolution for our readout. </br>1.2)ssrA Tag</br>To make proteins susceptible to degradation by the ClpXP protease you need to fuse (Ec)ssrA to the C-terminus of the protein. Since the native ssrA tag does not enable degradation of the protein upon sspB induction we needed to use the mutated version (Ec)ssrA DAS+4. [see Ec ClpXP for details]. Fusing this tag to mCherry should enable the induction of its degradation when expression or activity of sspB is induced.</br>1.3) Chassis</br>Since native E. coli carry a copy of sspB on their chromosomal DNA they express this protein their selves. This should lead to a great increasing of background degradation of ssrA tagged proteins. Furthermore it may prevent any interference - and therefore inducible degradation - upon induction of a plasmid carried copy of sspB. Using a sspB deficient mutant (strain: w3110) should enable us to clearly achieve inducible degradation of our reporter protein mCherry and furthermore comparable expression values for mCherry-ssrA and mCherry.</br>1.4)Expression System</br>Proper knowledge of the epxression of the reporter is basis for any further results. Therefore we wanted to first of all investigate the expression of mCherry. We decided to use an inducible expression system to obtain a tunable system escpecially for investigations related to the upcoming project parts. Because of several reasons we finally decided to use an IPTG inducible mCherry expression system incorporating the pLac promoter. Even though this promoter is quite leaky this is not that much of importance since mCherry is not functional part of our project but only the reporter. So we designed two expression systems for mCherry: one in fusion with the ssrA tag and one without.</br></br>1.5) Experiments – Reporter Expression</br>We want to investigate the following questions:</br>1)Is mCherry-ssrA working properly?</br>Even though ssrA is only a 15 amino acid small peptide tag there could be the chance that it – even though this chance is very low - greatly influences folding and therefore function of mCherry leading f.e. to a not fluorescing mCherry. </br>2)Is there a difference in expression rates for mCherry and mCherry-ssrA?</br>3)Is mCherry-ssrA degraded</br>Investigating this hypothesis we want to want to perform the following experiments:</br>1)Is mCherry-ssrA working properly?</br>Transform a pLac expression system for mCherry-ssrA in any E. coli strain and examine whether red fluorescence occurs or not.</br>2)</br>Is there a difference in expression rates for mCherry and mCherry-ssrA?</br>Co-transform similar pLac expression constructs of mCherry and mCherry-ssrA and an expression system for the repressor LacI to sspB deficient strain and induction with various concentrations of IPTG. </br>3)Is mCherry-ssrA degraded?</br>Transform similar pLac expression systems for mCherry-ssrA and mCherry in a LacI expressing E. coli strain (f.e. XL1-Blue) and iduce transcription with various concentrations of IPTG. A lower expression value is expected for mCherry-ssrA due to degradation induced by chromosomal encoded sspB.</br></br>Expression construct – sspB induced degradation</br>As a first approach we want to test whether it is possible to induce the degradation of mCherry-ssrA through inducing expression of full length sspB. For that purpose we designed a expression system for sspB including the Arabinose inducible pBAD promoter system. We decided on this system because of its very low background activity. This should allow for any greater activity of sspB even at lower expression values.</br>Focussing on the previous points we have to investigate the following questions:</br>1)Is the inducible degradation system working in our hands?</br>The most likely reason when the system is not working is that there are promoter issues: either the promoter does not work properly or induction was not carried out optimal.</br>2)Is the degradation rate of mCherry-ssrA directly dependent of the concentration of sspB?</br>It is possible that high concentrations off sspB inhibit degradation due to competitive binding. Investigating this question would greatly help us designing a proper expression plasmid for our final system.</br>3)How much does the degradation rate depend on the expression level of mCherry-ssrA?</br>It is of particular importance to know what expression rate of mCherry-ssrA is needed for the best readout.</br>4)Is it possible to induce degradation in native E. coli (with a chromosomal sspB)?</br>This would enable the use of our system in native E. coli without the need for a sspB deficient mutant – making it more easy applicable</br></br>To investigate this questions we want to conduct the following experiments:</br>1)Is the inducible degradation system working in our hands</br>2)Is the degradation rate of mCherry-ssrA directly depending on the concentration of sspB?</br>Co-transform the IPTG inducible mCherry-ssrA expression plasmid and the Arabinose inducible sspB plasmid to a sspB deficient strain. Conduct an assay with varying combinations of Arabinose and IPTG concentration.</br>3)How much does the degradation rate depend on the expression level of mCherry?</br>Same setup as for 1)</br>4)Is it possible to induce degradation in native E. coli (with a chromosomal sspB)?</br>Co-transform the IPTG inducible mCherry-ssrA expression plasmid and the Arabinose inducible sspB plasmid to an E.coli strain bearing chromosomal sspB. Conduct an assay with varying combinations of Arabinose and IPTG concentration.</br></br>Rapamycin inducible degradation with a split (Ec)sspB </br>For our final system we need to use the split version of sspB to enable induction of its activity upon heterodimerisation. The Rapamycin inducible sspB system designed by Davis et al offers a good way to test several parameters of the split system. It was characterized in a low to medium copy vector (pSB3C5).</br>We want to investigate the following questions:</br>1)Is the Rapamycin inducible degradation system working in our hands</br>Issues may occur when co-transforming the low to medium copy plasmid and the plac mCherry-ssrA expression plasmid. Furthermore</br>2)Is the system working when introduced in a high copy vector?</br>This would enable direct use of the part from the distribution plate. Furthermore it would be a very important step towards the question which expression levels are desired for our final system.</br>3)If the system is working in a high copy vector is there a difference for the optimal rapamycin concentration?";
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Revision as of 03:58, 5 October 2013
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