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Team:St Andrews/modelling
From 2011.igem.org
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<p class="textpart">We decided to utilise the pBAD strong promoter (<a href="http://partsregistry.org/Part:BBa_K206000">K206000</a>), which was created by the British Columbia iGEM 2009 team as a mutagenized form of the naturally occurring pBAD promoter. The modelling was based upon and used references pertaining to the pBAD promoter, also known as the 'ara operon'. pBAD and pBAD strong are both arabinose-inducible promoters.</p> | <p class="textpart">We decided to utilise the pBAD strong promoter (<a href="http://partsregistry.org/Part:BBa_K206000">K206000</a>), which was created by the British Columbia iGEM 2009 team as a mutagenized form of the naturally occurring pBAD promoter. The modelling was based upon and used references pertaining to the pBAD promoter, also known as the 'ara operon'. pBAD and pBAD strong are both arabinose-inducible promoters.</p> | ||
| - | <p class="textpart">In the absence and presence of arabinose, the pBAD promoter acts as a repressor and an inducer respectively. The gene products of pBAD in <i>Escherichia coli</i> allow the cells to take up and catabolize L-arabinose, a five-carbon sugar.</p> | + | <p class="textpart">In the absence and presence of arabinose, the pBAD promoter acts as a repressor and an inducer respectively. The gene products of pBAD in <i>Escherichia coli</i> allow the cells to take up and catabolize L-arabinose, a five-carbon sugar. pBAD’s very basic structure is depicted below:</p> |
| - | + | FIGURE 1 | |
<p class="textpart">The three enzymes that comprise the pBAD promoter cause the catabolism of the sugar arabinose as follows:</p> | <p class="textpart">The three enzymes that comprise the pBAD promoter cause the catabolism of the sugar arabinose as follows:</p> | ||
| - | + | araA – arabinose isomerase which converts arabinose to ribulose</p> | |
| - | + | araB – ribulokinase which phosphorylates ribulose</p> | |
| - | + | araD – ribulose-5-phosphate epimerase which converts ribulose-5-phosphate which can then be metabolised via the pentose phosphate pathway (Bharat Patel, http://trishul.ict.griffith.edu.au/courses/3011bbs/Lecture4.pdf)</p> | |
<p class="textpart">Adjacent to the 3 pBAD structural genes, there is the AraC regulatory gene. The dimeric (the compound comprises of two structurally similar subunits called monomers which together make a dimer (http://en.wikipedia.org/wiki/Dimer_(chemistry)). AraC protein actively represses its own synthesis as well as the synthesis of the pBAD genes, this occurs when arabinose is not present in the environment. The AraC protein binds to the half sites araO2 and ara I1 which creates a DNA loop, thus blocking the RNA polymerase from the pC and pBAD promoters. This can be seen in Figure 2, where it’s possible to comprehend the position of the relative half-sites.</p> | <p class="textpart">Adjacent to the 3 pBAD structural genes, there is the AraC regulatory gene. The dimeric (the compound comprises of two structurally similar subunits called monomers which together make a dimer (http://en.wikipedia.org/wiki/Dimer_(chemistry)). AraC protein actively represses its own synthesis as well as the synthesis of the pBAD genes, this occurs when arabinose is not present in the environment. The AraC protein binds to the half sites araO2 and ara I1 which creates a DNA loop, thus blocking the RNA polymerase from the pC and pBAD promoters. This can be seen in Figure 2, where it’s possible to comprehend the position of the relative half-sites.</p> | ||
| + | |||
| + | FIGURE 2 | ||
| + | |||
| + | <p class="textpart">When arabinose is present, it binds to the AraC protein and this destabilises the AraC protein binding to the araI1-O2 half-site looped complex, but stabilizes binding to the adjacent half-sites araI1 and araI2, which are upstream of the pBAD promoter. This then ‘straightens’ the DNA loop, (Carra and Schleif, ref to variation of half-site, 1993) allowing the activation of the transcription of pBAD. (Robert Schleif homepage)</p> | ||
Revision as of 08:36, 20 September 2011
Modelling
Promoter pBAD Strong
We decided to utilise the pBAD strong promoter (K206000), which was created by the British Columbia iGEM 2009 team as a mutagenized form of the naturally occurring pBAD promoter. The modelling was based upon and used references pertaining to the pBAD promoter, also known as the 'ara operon'. pBAD and pBAD strong are both arabinose-inducible promoters.
In the absence and presence of arabinose, the pBAD promoter acts as a repressor and an inducer respectively. The gene products of pBAD in Escherichia coli allow the cells to take up and catabolize L-arabinose, a five-carbon sugar. pBAD’s very basic structure is depicted below:
FIGURE 1The three enzymes that comprise the pBAD promoter cause the catabolism of the sugar arabinose as follows:
araA – arabinose isomerase which converts arabinose to ribulose araB – ribulokinase which phosphorylates ribulose araD – ribulose-5-phosphate epimerase which converts ribulose-5-phosphate which can then be metabolised via the pentose phosphate pathway (Bharat Patel, http://trishul.ict.griffith.edu.au/courses/3011bbs/Lecture4.pdf)Adjacent to the 3 pBAD structural genes, there is the AraC regulatory gene. The dimeric (the compound comprises of two structurally similar subunits called monomers which together make a dimer (http://en.wikipedia.org/wiki/Dimer_(chemistry)). AraC protein actively represses its own synthesis as well as the synthesis of the pBAD genes, this occurs when arabinose is not present in the environment. The AraC protein binds to the half sites araO2 and ara I1 which creates a DNA loop, thus blocking the RNA polymerase from the pC and pBAD promoters. This can be seen in Figure 2, where it’s possible to comprehend the position of the relative half-sites.
FIGURE 2When arabinose is present, it binds to the AraC protein and this destabilises the AraC protein binding to the araI1-O2 half-site looped complex, but stabilizes binding to the adjacent half-sites araI1 and araI2, which are upstream of the pBAD promoter. This then ‘straightens’ the DNA loop, (Carra and Schleif, ref to variation of half-site, 1993) allowing the activation of the transcription of pBAD. (Robert Schleif homepage)
