Team:Imperial College London/Wetlab/BioBricks

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=Submitted BioBricks=
=Submitted BioBricks=
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Imperial College iGEM 2009's uploaded BioBricks section on the Parts Registry can be found [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Imperial%20College%20London here].<br><br>
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===Summary Table of BioBricks Designed===
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200012 </partinfo>
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| Regulatory + | Composite
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[[Image:II09_Regulatory.png|40px]]
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| '''Lambda promoter (cIts responsive) ''' is different from the common lambda promoter in that it is able to be repressed by the temperature sensitive cI protein (BBa_K200011). When it is not being repressed after 42°C induction, it acts as a strong promoter.
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| <partinfo>BBa_K200012 </partinfo>
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| <partinfo>BBa_K200017 </partinfo>
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| Regulatory
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| Composite
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[[Image:II09_Regulatory.png|40px]]
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[[Image:II09_Composite.png|40px]]
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| '''Lambda promoter (cIts responsive)  ''' is different from the common lambda promoter in that it is able to be repressed by the temperature sensitive cI protein (BBa_K200011). When it is not being repressed after 42°C induction, it acts as a strong promoter.
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| '''RBS+OtsB''' This is an intermediate construct comprising a RBS upstream of the second enzyme for the production of trehalose. A functional transcriptional unit will be obtained upon the ligation of a promoter and the first enzyme (OtsA) upstream of the RBS. To close the unit a double terminator would be required downstream of the OtsB gene.
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| <partinfo>BBa_K200018 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''pCstA+RBS+GFP+TT''' This part contains a GFP (green fluorescent protein) reporter in the functional transcriptional unit. As such, activity of the pCstA promoter can be assessed by the expression of GFP.
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200019 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''pLacI+RBS+RFP+TT+pCstA+RBS+GFP+TT''' This is our testing construct to assess the two inducible promoters pLacI and pCstA through the expression of reporter molecules RFP and GFP.
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200020 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''pCstA+RBS''' This is an intermediate construct that can be ligated to a coding region and terminator to produce a functional transcriptional unit.
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200021 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''pLacI+RBS''' This is an intermediate construct that can be ligated to a coding region and terminator to produce a functional transcriptional unit.
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200022 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''Heat-inducible system with GFP reporter''' Using the BioBrick part by Harvard '08 ([http://partsregistry.org/Part:BBa_K098995 BBa_K098995]). We have shown BBa_K098995 to be [https://2009.igem.org/Team:Imperial_College_London/Achievements functional] and have improved its characterisation through successful testing with this construct containing a GFP reporter.
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200023 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''PAH+TT''' Ligation to a double terminator means that a closed transcriptional unit is produced once a promoter and RBS are ligated before this construct
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200024 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''RcsB+TT''' Colanic acid producing gene (RcsB) attached to a double terminator. This is an intermediate construct assembled just prior to promoter ligation.
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200025 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''pCstA+RBS+RcsB+TT''' This is the functional transcriptional unit which allows expression of the RcsB receiver protein and positive regulation of colanic acid production.
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200026 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''RcsB+RBS+GFP+TT''' This intermediate construct contains a GFP reporter to and can be used to assess the activity of the RcsB receiver protein. Different promoters can be attached upstream of the RBS to complete this transcriptional unit.
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200027 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''PAH+RBS+GFP+TT''' (Info here)
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200028 </partinfo>
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| Coding
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[[Image:II09_Coding.png|40px]]
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| '''Protease resistant PAH''' This protease resistant PAH bears a mutation that changes serine 16 into glutamine. This has the effect of mimicking phosphorylation of the protein; modification which has been shown to be associated with protection against proteases ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1216923/ Døskeland <i>et al.</i>, 1996])
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K200029 </partinfo>
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| Composite
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[[Image:II09_Composite.png|40px]]
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| '''pLacI+RBS+RcsB+TT''' This is the functional transcriptional unit which allows expression of the RcsB receiver protein and positive regulation of colanic acid production. It differs from BBa_K200025, above, by having a different promoter. The differences in functionality between these two constructs will be assessed.
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<a href="https://2009.igem.org/Team:Imperial_College_London/Major_results"><img width=150px src="https://static.igem.org/mediawiki/2009/0/0a/II09_POArrowLeft.png"></a>
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<a href="https://2009.igem.org/Team:Imperial_College_London/Achievements"><img width=150px src="https://static.igem.org/mediawiki/2009/2/2d/II09_POArrowRight.png"></a></center>
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Latest revision as of 02:27, 22 October 2009

Submitted BioBricks



Summary Table of BioBricks Designed

Registry Code Type Sequence Description
Coding

II09 Coding.png

RcsB is a receiver protein which acts as a positive regulator of a number of genes including capsule genes responsible for colanic acid production.
Coding

II09 Coding.png

Dam (DNA Adenine Methylase) The methylase encoded by the dam gene ([http://en.wikipedia.org/wiki/Dam_(methylase) Dam methylase]) transfers a methyl group from S-adenosylmethionine to the N6 position of the adenine residues in the sequence GATC, this protects the DNA from cleavage.
Coding

II09 Coding.png

Colanic acid global regulator ygiV (B3023) increases the production of colanic acid further in conjunction with RcsB by acting as a repressor for mcbR/yncC promoter. YncC/mcbR normally repress colanic acid overproduction so as to increase biofilm formation.
Coding

II09 Coding.png

Waal Ligase is an enzyme responsible for the ligation of an [http://en.wikipedia.org/wiki/O_antigen#O-antigen O-antigen] to the core [http://en.wikipedia.org/wiki/Oligosaccharide oligosaccharide] in the Gram-negative bacterium's outer membrane.
Coding

II09 Coding.png

OtsA is the first of two required in the conversion of glucose to [http://en.wikipedia.org/wiki/Trehalose trehalose].

This enzyme catalyses the following reaction:

UDP-glucose + D-glucose 6-phosphate -> UDP + alpha,alpha-trehalose 6-phosphate

Coding

II09 Coding.png

OtsB This enzyme is the second of two required for the conversion of glucose to [http://en.wikipedia.org/wiki/Trehalose trehalose].

This enzyme catalyses the following reaction:

alpha,alpha-trehalose 6-phosphate + H2O -> alpha,alpha-trehalose + phosphate

Coding

II09 Coding.png

[http://en.wikipedia.org/wiki/Cellulase Cellulase] mainly catalyses the reactions that changes crystalline [http://en.wikipedia.org/wiki/Cellulose cellulose] to [http://en.wikipedia.org/wiki/Cellobiose cellobiose] and then finally to glucose. This cellulase is protease resistant.
Coding

II09 Coding.png

[http://en.wikipedia.org/wiki/Phenylalanine_hydroxylase Phenylalanine hydroxylase] is the enzyme that breaks down [http://en.wikipedia.org/wiki/Phenylalanine phenylalanine] to [http://en.wikipedia.org/wiki/Tyrosine tyrosine]. Deficiency of this enzyme activity results in the autosomal recessive disorder [http://en.wikipedia.org/wiki/Phenylketonuria phenylketonuria].
Coding

II09 Coding.png

[http://en.wikipedia.org/wiki/Opiorphin Opiorphin] is a pentapeptide that inhibits the breakdown of endorphines. This results in powerful painrelief. This part contains an enterokinase cleavage site to facilitate synthesis and subsequent activation.
Coding

II09 Coding.png

[http://en.wikipedia.org/wiki/Opiorphin Opiorphin] is a pentapeptide that inhibits the breakdown of endorphines. This results in powerful painrelief. This part contains an enterokinase cleavage site to facilitate synthesis and subsequent activation. This part also contains a HIS tag to facilitate high quality purification.
Composite

II09 Composite.png

Restriction enzyme [http://www.thelabrat.com/restriction/DpnII.shtml DpnII] is a Type II restriction enzyme that recognises the sequence GATC. Its activity can be blocked by dam methylation.
Composite

II09 Composite.png

Restriction enzyme [http://www.thelabrat.com/restriction/TaqI.shtml TaqI] is a Type II restriction enzyme that recognises the sequence TCGA. Its activity can be blocked by dam methylation.
Composite

II09 Composite.png

This Lamda cI repressor has a cI857 mutation that results in denaturation of the repressor when the temperature is raised from 30 to 42°C, thereby allowing lambda promoter expression.

When the temperature is raised, typically to 42°C, the functionality of the protein is lost and the cI repressor is no longer able to bind to the operators on its promoter. Therefore, lambda promoter expression increases.

Composite

II09 Regulatory.png

Lambda promoter (cIts responsive) is different from the common lambda promoter in that it is able to be repressed by the temperature sensitive cI protein (BBa_K200011). When it is not being repressed after 42°C induction, it acts as a strong promoter.
Composite

II09 Composite.png

RBS+OtsB This is an intermediate construct comprising a RBS upstream of the second enzyme for the production of trehalose. A functional transcriptional unit will be obtained upon the ligation of a promoter and the first enzyme (OtsA) upstream of the RBS. To close the unit a double terminator would be required downstream of the OtsB gene.
Composite

II09 Composite.png

pCstA+RBS+GFP+TT This part contains a GFP (green fluorescent protein) reporter in the functional transcriptional unit. As such, activity of the pCstA promoter can be assessed by the expression of GFP.
Composite

II09 Composite.png

pLacI+RBS+RFP+TT+pCstA+RBS+GFP+TT This is our testing construct to assess the two inducible promoters pLacI and pCstA through the expression of reporter molecules RFP and GFP.
Composite

II09 Composite.png

pCstA+RBS This is an intermediate construct that can be ligated to a coding region and terminator to produce a functional transcriptional unit.
Composite

II09 Composite.png

pLacI+RBS This is an intermediate construct that can be ligated to a coding region and terminator to produce a functional transcriptional unit.
Composite

II09 Composite.png

Heat-inducible system with GFP reporter Using the BioBrick part by Harvard '08 ([http://partsregistry.org/Part:BBa_K098995 BBa_K098995]). We have shown BBa_K098995 to be functional and have improved its characterisation through successful testing with this construct containing a GFP reporter.
Composite

II09 Composite.png

PAH+TT Ligation to a double terminator means that a closed transcriptional unit is produced once a promoter and RBS are ligated before this construct
Composite

II09 Composite.png

RcsB+TT Colanic acid producing gene (RcsB) attached to a double terminator. This is an intermediate construct assembled just prior to promoter ligation.
Composite

II09 Composite.png

pCstA+RBS+RcsB+TT This is the functional transcriptional unit which allows expression of the RcsB receiver protein and positive regulation of colanic acid production.
Composite

II09 Composite.png

RcsB+RBS+GFP+TT This intermediate construct contains a GFP reporter to and can be used to assess the activity of the RcsB receiver protein. Different promoters can be attached upstream of the RBS to complete this transcriptional unit.
Composite

II09 Composite.png

PAH+RBS+GFP+TT (Info here)
Coding

II09 Coding.png

Protease resistant PAH This protease resistant PAH bears a mutation that changes serine 16 into glutamine. This has the effect of mimicking phosphorylation of the protein; modification which has been shown to be associated with protection against proteases ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1216923/ Døskeland et al., 1996])
Composite

II09 Composite.png

pLacI+RBS+RcsB+TT This is the functional transcriptional unit which allows expression of the RcsB receiver protein and positive regulation of colanic acid production. It differs from BBa_K200025, above, by having a different promoter. The differences in functionality between these two constructs will be assessed.



Mr. Gene   Geneart   Clontech   Giant Microbes

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