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Team:UPO-Sevilla/Project/Improving Flip Flop/Results/Plasmids and controls
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<h1>Plasmids and controls</h1> | <h1>Plasmids and controls</h1> | ||
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| + | <h2>Control construction</h2> | ||
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<p>The improved flip-flop was designed to make the control construction an easier task. By digesting the devices with the correct enzymes, we could remove or add any part anywhere within the improve flip-flop module I. The necessary control for the improved flip-flop requires two changes in the device structure: (a) removing the SspB gene from the module I to achieve a flip flop without protease activity, and (b) using an empty plasmid which provide the antibiotic resistance cassette and its possibly secondary effects instead of the plasmid with the improved flip-flop module II. This control was constructed, but analytic digestions to verify the new device are still in progress. </p> | <p>The improved flip-flop was designed to make the control construction an easier task. By digesting the devices with the correct enzymes, we could remove or add any part anywhere within the improve flip-flop module I. The necessary control for the improved flip-flop requires two changes in the device structure: (a) removing the SspB gene from the module I to achieve a flip flop without protease activity, and (b) using an empty plasmid which provide the antibiotic resistance cassette and its possibly secondary effects instead of the plasmid with the improved flip-flop module II. This control was constructed, but analytic digestions to verify the new device are still in progress. </p> | ||
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| + | <div class="center"> | ||
| + | <img width="675px" src="https://static.igem.org/mediawiki/2011/3/3e/UPOSevillaTEAM2011.Control.jpg" alt="Control Construction" /> | ||
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| + | <h2>Vectors for improved flip-flop expression</h2> | ||
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<p>The copy number of plasmids results to be an important factor for the function of regulatory circuits as flip-flops. Thus, we performed our fluorometry measurements with the improved flip-flop module II (asRNA) in high and low copy number plasmids. The asRNA module was cloned in a high copy number pSB1T3 vector and in a low copy pSB4A5 vector. The improved flip-flop module I was used in a medium copy number pUC commercial vector. Also, this module was integrated in the chromosome of the X90 SspB- and RybB- <em>E. coli</em> strain in single copy by using the miniTn7BB-Gm functions, but the data of these measurements are too preliminary to be shown.</p> | <p>The copy number of plasmids results to be an important factor for the function of regulatory circuits as flip-flops. Thus, we performed our fluorometry measurements with the improved flip-flop module II (asRNA) in high and low copy number plasmids. The asRNA module was cloned in a high copy number pSB1T3 vector and in a low copy pSB4A5 vector. The improved flip-flop module I was used in a medium copy number pUC commercial vector. Also, this module was integrated in the chromosome of the X90 SspB- and RybB- <em>E. coli</em> strain in single copy by using the miniTn7BB-Gm functions, but the data of these measurements are too preliminary to be shown.</p> | ||
Revision as of 01:12, 29 October 2011
Plasmids and controls
Control construction
The improved flip-flop was designed to make the control construction an easier task. By digesting the devices with the correct enzymes, we could remove or add any part anywhere within the improve flip-flop module I. The necessary control for the improved flip-flop requires two changes in the device structure: (a) removing the SspB gene from the module I to achieve a flip flop without protease activity, and (b) using an empty plasmid which provide the antibiotic resistance cassette and its possibly secondary effects instead of the plasmid with the improved flip-flop module II. This control was constructed, but analytic digestions to verify the new device are still in progress.
Vectors for improved flip-flop expression
The copy number of plasmids results to be an important factor for the function of regulatory circuits as flip-flops. Thus, we performed our fluorometry measurements with the improved flip-flop module II (asRNA) in high and low copy number plasmids. The asRNA module was cloned in a high copy number pSB1T3 vector and in a low copy pSB4A5 vector. The improved flip-flop module I was used in a medium copy number pUC commercial vector. Also, this module was integrated in the chromosome of the X90 SspB- and RybB- E. coli strain in single copy by using the miniTn7BB-Gm functions, but the data of these measurements are too preliminary to be shown.
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