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<figcaption><b>Fig.2: BFP expression for 3 isolated of each of the 5 parts. BBa_K3174002 and BBa_K3174003 have the lowest BFP expression levels because they have mutated as a result of high metabolic stress. BBa_K3174004 has the highest BFP expression level because it is stable. BBa_K3174006 and BBa_K3174007 have intermediate BFP expression levels. </figcaption>
 
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Revision as of 22:50, 15 October 2019


Blue Fluorescent Protein with Strong Promoter and Strong RBS

This part has the blue fluorescent protein (mTagBFP) gene inserted downstream of a strong promoter and strong ribosome binding site.

BBa_J23104 is part of the Anderson Series of constitutive promoters designed by iGEM2006_Berkeley.

BBa_B0034 is an RBS based on the Elowitz repressilator and was designed in 2003. Its relative strength was characterized by IIT Madras in 2016 and by Team Warsaw in 2010.

BBa_K592100 is a blue fluorescent protein designed by iGEM11_Uppsala-Sweden in 2011. It has a maximum emission of 456 nm.

Fig.1: Differential growth rates of 5 BFP plasmids in TOP10 cells: (left to right) BBa_K3174002, BBa_K3174003, BBa_K3174004, BBa_K3174006, and BBa_K3174007. Single colonies are scarce for BBa_K3174002 and BBa_K3174003 , more abundant for BBa_K3174004, and most abundant for BBa_K3174006 and BBa_K3174007. This differential growth rate is due to the different levels of metabolic burden each strain expresses due to their relative promoter and RBS strengths.

The differential growth rate observed between these 5 parts results from different promoter and RBS combinations that each impose a proportional level of metabolic burden on the host cell. BBa_K3174002 and BBa_K3174003 both have strong promoters, therefore, they grow very few single colonies when streaked on a plate and express the lowest level of BFP because they break very easily. For this reason, they should be used with caution because the burdened cells are quickly outcompeted by the more fit mutated cells which contain broken genetic circuits. BBa_K3174004 grows more single colonies than BBa_K3174002 and BBa_K3174003 and expresses BFP at the highest level of these 5 parts because it is the most stable, containing a strong promoter, and weak RBS. BBa_K3174006 and BBa_K3174007 grow the most single colonies and express BFP at a higher average level than BBa_K3174002 and BBa_K3174003 but at a lower level than BBa_K3174004 because they both have medium promoters. BBa_K31740064 seems to be the most stable of these 5 parts and is recommended for experiments that rely on evolutionary stability.

Fig.2: BFP expression for 3 isolated of each of the 5 parts. BBa_K3174002 and BBa_K3174003 have the lowest BFP expression levels because they have mutated as a result of high metabolic stress. BBa_K3174004 has the highest BFP expression level because it is stable. BBa_K3174006 and BBa_K3174007 have intermediate BFP expression levels.



Usage and Biology

UT Austin iGEM 2019 Team transformed this part into the E.coli DH10B burden monitor (https://2019.igem.org/Team:Austin_UTexas) and used it as a standard with which cellular burden could be quantified (by means of a GFP expression vs growth rate model) for other BioBricks that had been similarly transformed into the burden strain.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]