From 2009.igem.org

Submitted BioBricks

Summary Table of BioBricks Designed

Registry Code	Type	Sequence Description
	Coding	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	Dam (DNA Adenine Methylase) The methylase encoded by the dam gene (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	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	Waal Ligase is an enzyme responsible for the ligation of an O-antigen to the core oligosaccharide in the Gram-negative bacterium's outer membrane.
	Coding	OtsA is the first of two required in the conversion of glucose to trehalose. This enzyme catalyses the following reaction: UDP-glucose + D-glucose 6-phosphate -> UDP + alpha,alpha-trehalose 6-phosphate
	Coding	OtsB This enzyme is the second of two required for the conversion of glucose to trehalose. This enzyme catalyses the following reaction: alpha,alpha-trehalose 6-phosphate + H2O -> alpha,alpha-trehalose + phosphate
	Coding	Cellulase mainly catalyses the reactions that changes crystalline cellulose to cellobiose and then finally to glucose. This cellulase is protease resistant.
	Coding	Phenylalanine hydroxylase is the enzyme that breaks down phenylalanine to tyrosine. Deficiency of this enzyme activity results in the autosomal recessive disorder phenylketonuria.
	Coding	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	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	Restriction enzyme DpnII is a Type II restriction enzyme that recognises the sequence GATC. Its activity can be blocked by dam methylation.
	Composite	Restriction enzyme TaqI is a Type II restriction enzyme that recognises the sequence TCGA. Its activity can be blocked by dam methylation.
	Composite	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	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	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	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	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	pCstA+RBS This is an intermediate construct that can be ligated to a coding region and terminator to produce a functional transcriptional unit.
	Composite	pLacI+RBS This is an intermediate construct that can be ligated to a coding region and terminator to produce a functional transcriptional unit.
	Composite	Heat-inducible system with GFP reporter Using the BioBrick part by Harvard '08 (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	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	RcsB+TT Colanic acid producing gene (RcsB) attached to a double terminator. This is an intermediate construct assembled just prior to promoter ligation.
	Composite	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	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	PAH+RBS+GFP+TT (Info here)
	Coding	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 (Døskeland et al., 1996)
	Composite	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.

Team:Imperial College London/Wetlab/BioBricks

From 2009.igem.org

Submitted BioBricks

Summary Table of BioBricks Designed