Part:BBa_J04450

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 equal functionality in simulated microgravity (0g) as in the normal gravity of the Earth.
(--Valentijn 19:38, 19 October 2016 (UTC))

[http://2017.igem.org/Team:UChicago Team UChicago 2017] contributed to this part by improving/changing the documented sequence through mutagenesis to create blunt-end restriction sites for cloning not within the prefix/suffix region (created BBa_K2428000).
(--pzulueta97 21:14, 25 October 2017 (UTC) )

[http://2017.igem.org/Team:Grenoble-Alpes Team Grenoble-Alpes 2017] contributed to the characterisation of this part by testing the time of apparition of fluorescence, in presence of IPTG or not (because the promoter leaks), as well as they contributed to the improvement of this part by using its fluorescence as a detection signal to be able to detect Vibrio Cholerae.
(--NoreenLouis 20:47, 26 October 2017 (UTC) )

[http://2017.igem.org/Team:Kingsborough_NY Team Kingsborough NY 2017] contributed to the characterization of this part by showing decreased fluorescence when expressed either in a higher salt media - such as LB with 3% sodium chloride - or E. coli that lacks tmRNA, the principal component of the cell's ribosome rescue system. [http://2017.igem.org/Team:Kingsborough_NY/RFP See the data at our Wiki]
(--djcamenares 17:56, 27 October 2017 (UTC) )

[http://2017.igem.org/Team:iTesla-SoundBio Team iTesla SoundBio 2017] contributed to the characterization of this part by analyzing the rate of false positives when using the coloring of transformed colonies as a red/white screen for determining experimental success.
(--gladish 01:26, 28 October 2017 (UTC) )

Pictures

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  BBa_J04450 visualized under non-UV lightbox

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  BBa_J04450 visualized under 254nm wavelength UV lightbox

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  BBa_J04450 Colonies

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  Pantoea agglomerans colonies in chloramphenicol, containing BBa_J04450 in the pSB1C3 backbone

Team INSA-UPS France 2017 : usage in Vibrio harveyi strain engineered by conjugation

In the context of the iGEM INSA-UPS France project 2017, BBa_J04450 was tested in the Vibrio harveyi background. To the best of our knowledge, RFP has never been used in this strain. BBa_J04450 biobrick was cloned in a broad host range plasmid (pBBR1MCS-4) and conjugated into V. harveyi. The protocol of triparental mating can be found here. Its expression has been studied by fluoresence microscopy in Vibrio harveyi .

Monitoring of the production of RFP by V. harveyi strain microscopy Vibrio harveyi JMH626 conjugated with pBBR1MCS-4-RFP (panels C & D) or with pBBR1MCS-4 (control ; panels A & B) was analyzed by optic microscopy (panel A & C) or fluorescence microscopy (panels B & D).

The microscopy results demonstrated the fonctional production of RFP in Vibrio harveyi, and hence, the functionality of part BBa_J04450 in this background.

IIT Madras 2016's Characterization

Experimentation

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 [http://2016.igem.org/Team:IIT-Madras/Model#Noise_in_Devices| 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.

[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 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.

[Image:]