RFP Coding Device
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))
Team Leiden 2016 contributed to the characterisation of this part by showing equal functionality in simulated microgravity (0g) as in the normal gravity of the Earth.
(--Valentijn 19:38, 19 October 2016 (UTC))
Usage as a cloning tool
Team INSA UPS France 2017's usage in Vibrio harveyi strain engineered by conjugation
RFP gene which come from iGEM 2017 starting plate was used as reporter gene in several modules of the croc’n cholera. Thus its expression has been studied by microscopy in Vibrio harveyi .
RFP gene was cloned in a broad host range plasmid (pBBR1MCS4) and conjugated to V. harveyi. The protocol of triparental mating can be found (here)
The microscopy results revealed the production of RFP in Vibrio harveyi.
IIT Madras 2016's Characterization
This BioBrick was used along with various GFP producing devices to understand the role of RBS and Promoter parts in giving rise to intrinsic noise in E. coli DH5alpha. Expression data for GFP and RFP proteins were obtained using flow cytometry (BD FACS Aria III) at 3hr, 6hr, 9hr and 12hr stage of growth along with cells expressing only GFP, only RFP and none. Cumulative intrinsic and extrinsic noise were measured using modified Elowitz formula. OD600 values for specific growth rate estimation were obtained using Spectrophotometer over an interval of an hour for 12 hours. Given specific growth rates are in it's logarithmic values. This BioBrick can be used to characterize noise and strength of complex devices by cloning this device with given device, which produces a different reporter protein. In graphs, we have R11-B32, R11-B34, J14-B3, J17-B34, R11-B30 and R11-B31 in pSB1A2 plasmid backbone.
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.
Usage in Chromobacterium Violaceum
Team Tec-Monterrey 2016 characterized the output of the part BBa_J04450 in a novel chassis, Chromobacterium Violaceum, as it produces a native purple pigment Violacein, we were curious whether RFP would be useful as a reporter gene. Furthermore, we characterized its expression under lac promoter. We did the transformation of C. Violaceum by a method that has not been reported yet, we made C. Violaceum competent cells with the protocol that is in our wiki, we concluded that the best O.D. for the heat shock transformation is 0.5 since it showed clearly better results than 0.4 or 0.6, we will continue to work in the transformation efficiency.