Difference between revisions of "Part:BBa J04450"
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[[Image:co-inef-gn.jpg|200px|thumb|left|An inefficient assembly ligation of two BioBricks into the pSB1C3 backbone producing many red colonies.]][[Image:co-ef-gn.jpg|200px|thumb|left|A more efficient, single BioBrick ligation into the pSB1C3 backbone.]]
 
[[Image:co-inef-gn.jpg|200px|thumb|left|An inefficient assembly ligation of two BioBricks into the pSB1C3 backbone producing many red colonies.]][[Image:co-ef-gn.jpg|200px|thumb|left|A more efficient, single BioBrick ligation into the pSB1C3 backbone.]]
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==Usage in Chromobacterium Violaceum==
 
==Usage in Chromobacterium Violaceum==
  
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[http://2016.igem.org/Team:Tec-Monterrey] characterized the output of this part in a novel chassis, Chromobacterium Violaceum, as it produces a native purple pigment Violaceum, we were curious wether RFP would be useful as a reporter gene. Furthermore, we characterized its expression under lac promoter. We were unable to confirm wether or not C. Violaceum had the lac operon, BLAST analysis of its genome suggested that C. Violaceum did not have this regulating system; our characterization demostrates that lac promoter can be used as a constitutive promoter.
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[http://2016.igem.org/Team:Tec-Monterrey Team Tec-Monterrey 2016] characterized the output of this part in a novel chassis, Chromobacterium Violaceum, as it produces a native purple pigment Violaceum, we were curious wether RFP would be useful as a reporter gene. Furthermore, we characterized its expression under lac promoter. We were unable to confirm wether or not C. Violaceum had the lac operon, BLAST analysis of its genome suggested that it did not have this regulating system; our characterization demostrates that the lac promoter can be used as a constitutive promoter in this chassis.

Revision as of 05:55, 18 October 2016


RFP Coding Device

LacI
R0010
B0034		mRFP1
E1010
B0015

The colonies are clearly red in color under natural light after about 18 hours. Smaller colonies are visibly red under UV. The RFP part does not contain a degradation tag and the RBS is strong.

  • LacI sensitive
  • CAP sensitive

This part is commonly used, but can fail if the system contains LacI or CAP protein.
(--Meagan 15:39, 23 July 2009 (UTC))

[http://2012.igem.org/Team:TU_Munich Team TU_Munich 2012] improved this part by making it compatible to RFC10 and RFC25 (see: BBa_K801100)
(--VolkerMorath 15:02, 21 October 2012 (UTC))

[http://2013.igem.org/Team:NRP-UEA-Norwich Team NRP-UEA 2013] improved this part by adding a NdeI restriction site before the RFP gene. (see: BBa_K1041000)
(--holusac 20:46, 14 August 2013 (UTC))

[http://2015.igem.org/Team:Warwick Team Warwick 2015] improved this part by analysing the effect of copy number on gene expression.
(--Lcarroll 20:48, 25 September 2015 (UTC))

[http://2016.igem.org/Team:Leiden Team Leiden 2016] contributed to the characterisation of this part by showing functions in simulated microgravity and Martian gravity.
(--Valentijn 12:55, 14 October 2016 (UTC))

Pictures

  • BBa_J04450 visualized under non-UV lightbox

  • BBa_J04450 visualized under 254nm wavelength UV lightbox

  • BBa_J04450 Colonies

Usage as a cloning tool

[http://2010.igem.org/Team:Groningen Team Groningen 2010] reports the usage of this part as a cloning tool. When ligating any part, or part assembly, into any standard backbone that contains this part, the non-restricted and single-restricted backbones that self-circularize will produce red colonies on rich media plates (we use TY). These undesired transformants can than be avoided in the screening for the correct construct. With this method, the backbone desired for a new construct does not need to be purified from agarose gel to decrease the amount of undesired tranformants caused by ligation of the original part present in the backbone. The amount of incorrect transformants depends, of course, on the ratio of backbone (mixed with J04450) vs. BioBrick insert, the size of the BioBrick insert, and whether the insert is an assembly of two BioBricks. The images below show two ligations with different efficiencies.

An inefficient assembly ligation of two BioBricks into the pSB1C3 backbone producing many red colonies.
A more efficient, single BioBrick ligation into the pSB1C3 backbone.



Usage in Chromobacterium Violaceum

[http://2016.igem.org/Team:Tec-Monterrey Team Tec-Monterrey 2016] characterized the output of this part in a novel chassis, Chromobacterium Violaceum, as it produces a native purple pigment Violaceum, we were curious wether RFP would be useful as a reporter gene. Furthermore, we characterized its expression under lac promoter. We were unable to confirm wether or not C. Violaceum had the lac operon, BLAST analysis of its genome suggested that it did not have this regulating system; our characterization demostrates that the lac promoter can be used as a constitutive promoter in this chassis.