Team:Virginia Commonwealth/Design

From 2009.igem.org

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==UP-element design==
==UP-element design==
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Work has been done to identify a consensus UP element on some promoters which interacts with an alpha subunit on RNAP (Estrem ST, Gaal T, Ross W, Gourse RL, Identification of an UP element consensus sequence for bacterial promoters, ''PNAS'', (1998), 95, 9761-9766.). This UP element has been shown to significantly increase the RNA polymerase-recruiting power of promoters and is, therefore, a transcriptional enhancer.
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UP-elements are known to significantly increase the RNA polymerase-recruiting power of promoters by interacting with the alpha subunit of RNA polymerase. Work has been done to identify a consensus sequence for this transcriptional enhancer based on 31 natural ''E. coli'' promoters (Estrem ST, Gaal T, Ross W, Gourse RL, Identification of an UP element consensus sequence for bacterial promoters, ''PNAS'', (1998), 95, 9761-9766.).
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[[Image:VCU 2009 tabulated UP-elements.png|thumb|center|400px|Tabulation of UP-elements in ''E. coli'' and nucleotide frequency at each position (adapted from Estrem ST, Gaal T, Ross W, Gourse RL, Identification of an UP element consensus sequence for bacterial promoters, ''PNAS'', (1998), 95, 9761-9766.)]]
[[Image:VCU 2009 tabulated UP-elements.png|thumb|center|400px|Tabulation of UP-elements in ''E. coli'' and nucleotide frequency at each position (adapted from Estrem ST, Gaal T, Ross W, Gourse RL, Identification of an UP element consensus sequence for bacterial promoters, ''PNAS'', (1998), 95, 9761-9766.)]]
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We are working on creating this UP element as a modular BioBrick part and characterizing its activity using the promoter characterization method that we develop.  We will also manipulate this element using a bottom-up design approach to get UP elements of varying degree's of strength.  Thus increasing the level of control we have over gene expression.
We are working on creating this UP element as a modular BioBrick part and characterizing its activity using the promoter characterization method that we develop.  We will also manipulate this element using a bottom-up design approach to get UP elements of varying degree's of strength.  Thus increasing the level of control we have over gene expression.
[[Image:VCU 2009 UP-element tabulated nucleotide frequency.png|thumb|center|650px|Tabulation of UP-element sequences in order of observed strengths and correlation with nucleotide frequency]]
[[Image:VCU 2009 UP-element tabulated nucleotide frequency.png|thumb|center|650px|Tabulation of UP-element sequences in order of observed strengths and correlation with nucleotide frequency]]
[[Image:VCU 2009 UP-element variable regions.png|thumb|center|650px|UP-element consensus sequence and observed variable regions for selected sequences (that most closely matched the consensus sequence)]]
[[Image:VCU 2009 UP-element variable regions.png|thumb|center|650px|UP-element consensus sequence and observed variable regions for selected sequences (that most closely matched the consensus sequence)]]
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[[Image:VCU 2009 UP-element design sequences.png|thumb|center|930px|Sequences of designed UP-elements]]
===Design strategies===
===Design strategies===
Promoter design will be approached from the bottom up.  First, a consensus promoter sequence will be studied and accepted by researching the work done by others on various promoters.  This consensus will be our starting point as we identify which nucleotide and nucleotide sequences are most important to RNAP binding affinity.  We will use direct synthesis to construct promoter sequences that we will test.
Promoter design will be approached from the bottom up.  First, a consensus promoter sequence will be studied and accepted by researching the work done by others on various promoters.  This consensus will be our starting point as we identify which nucleotide and nucleotide sequences are most important to RNAP binding affinity.  We will use direct synthesis to construct promoter sequences that we will test.
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==Future work==
==Future work==
*Constitutive promoters
*Constitutive promoters

Revision as of 16:45, 21 October 2009


UP-element design

UP-elements are known to significantly increase the RNA polymerase-recruiting power of promoters by interacting with the alpha subunit of RNA polymerase. Work has been done to identify a consensus sequence for this transcriptional enhancer based on 31 natural E. coli promoters (Estrem ST, Gaal T, Ross W, Gourse RL, Identification of an UP element consensus sequence for bacterial promoters, PNAS, (1998), 95, 9761-9766.).

Tabulation of UP-elements in E. coli and nucleotide frequency at each position (adapted from Estrem ST, Gaal T, Ross W, Gourse RL, Identification of an UP element consensus sequence for bacterial promoters, PNAS, (1998), 95, 9761-9766.)


We are working on creating this UP element as a modular BioBrick part and characterizing its activity using the promoter characterization method that we develop. We will also manipulate this element using a bottom-up design approach to get UP elements of varying degree's of strength. Thus increasing the level of control we have over gene expression.

Tabulation of UP-element sequences in order of observed strengths and correlation with nucleotide frequency
UP-element consensus sequence and observed variable regions for selected sequences (that most closely matched the consensus sequence)
Sequences of designed UP-elements

Design strategies

Promoter design will be approached from the bottom up. First, a consensus promoter sequence will be studied and accepted by researching the work done by others on various promoters. This consensus will be our starting point as we identify which nucleotide and nucleotide sequences are most important to RNAP binding affinity. We will use direct synthesis to construct promoter sequences that we will test.

Future work

  • Constitutive promoters
    • There are several types of constitutive promoters that we can focus on. These promoters are separate in regard to their respective sigma binding domains. Constitutive promoters that interact with the sigma 70 domain of RNAP are most common most prevalent being as how sigma 70 RNAP is most common at normal cellular growth conditions. The alternatives would be sigma 54 and sigma 32 binding factors, these types of polymerases are more active when the cell is under certain strain, such as heat shock of nitrogen starvation.
    • There has already been considerable work done with sigma 70 RNAP including an established promoter library of varying promoter strengths available on the parts registry. We will develop and implement a method to fully characterize and standardize these promoters' activity and upload it to the registry.


  • T7 Promoters
    • T7 phage promoters have long been utilized as a way to create orthogonal gene expression. There is very little variation in the strength of T7 promoters available via the BioBrick registry. Our goal is to identify a consensus T7 promoter sequence and use our bottom-up design approach to create a library of T7 promoters which we will characterize and upload to the registry.