"
Team:Imperial College London/Wetlab/BioBricks
From 2009.igem.org
(Difference between revisions)
| (31 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
{{Imperial/09/TemplateTop}} | {{Imperial/09/TemplateTop}} | ||
| - | |||
=Submitted BioBricks= | =Submitted BioBricks= | ||
| - | + | ||
| + | <html><iframe src="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Imperial%20College%20London" width="100%" height="600px"></iframe></html> | ||
| + | <br><br> | ||
| + | ===Summary Table of BioBricks Designed=== | ||
<!--template:----------------------------------------------------------------------------> | <!--template:----------------------------------------------------------------------------> | ||
| Line 99: | Line 101: | ||
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| + | | <partinfo>BBa_K200012 </partinfo> | ||
| + | | Regulatory + | Composite | ||
| + | [[Image:II09_Regulatory.png|40px]] | ||
| + | | '''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. | ||
| + | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| <partinfo>BBa_K200017 </partinfo> | | <partinfo>BBa_K200017 </partinfo> | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | '''RBS+OtsB''' ( | + | | '''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. |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 110: | Line 117: | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | '''pCstA+RBS+GFP+TT''' ( | + | | '''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. |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 117: | Line 124: | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | '''pLacI+RBS+RFP+TT+pCstA+RBS+GFP+TT''' | + | | '''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. |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 124: | Line 131: | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | '''pCstA+RBS''' | + | | '''pCstA+RBS''' This is an intermediate construct that can be ligated to a coding region and terminator to produce a functional transcriptional unit. |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 131: | Line 138: | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | '''pLacI+RBS''' | + | | '''pLacI+RBS''' This is an intermediate construct that can be ligated to a coding region and terminator to produce a functional transcriptional unit. |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 138: | Line 145: | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | ''' | + | | '''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. |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 145: | Line 152: | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | '''PAH+TT''' | + | | '''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 |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 152: | Line 159: | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | '''RcsB+TT''' ( | + | | '''RcsB+TT''' Colanic acid producing gene (RcsB) attached to a double terminator. This is an intermediate construct assembled just prior to promoter ligation. |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 159: | Line 166: | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | '''pCstA+RBS+RcsB+TT''' | + | | '''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. |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 166: | Line 173: | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | '''RcsB+RBS+GFP+TT''' | + | | '''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. |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 180: | Line 187: | ||
| Coding | | Coding | ||
[[Image:II09_Coding.png|40px]] | [[Image:II09_Coding.png|40px]] | ||
| - | | '''Protease resistant PAH''' ( | + | | '''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]) |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| Line 187: | Line 194: | ||
| Composite | | Composite | ||
[[Image:II09_Composite.png|40px]] | [[Image:II09_Composite.png|40px]] | ||
| - | | '''pLacI+RBS+RcsB+TT''' | + | | '''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. |
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | |- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1" | ||
| - | |||
|} | |} | ||
| - | + | <br> | |
| + | <html><center> | ||
| + | <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> | ||
| + | <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> | ||
| + | </html> | ||
| + | <br> | ||
{{Imperial/09/TemplateBottom}} | {{Imperial/09/TemplateBottom}} | ||
Latest revision as of 02:27, 22 October 2009
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 ([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
| 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 [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
| 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
| 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
| [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
| [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
| [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
| [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
| 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
| 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
| 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 ([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
| 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 ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1216923/ 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. |
