Part:BBa_K3174002

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: 5 different BFP plasmids in TOP10 cells grown in a 5mL O/N culture viewed under a UV light: (left to right) BBa_K3174002, BBa_K3174003, BBa_K3174004, BBa_K3174006, and BBa_K3174007. The same streaking method was used for all 5 of these plates. BBa_K3174002 and BBa_K3174003 have very small colonies. BBa_K3174004, and many for BBa_K3174006 and BBa_K3174007. The disparity in growth rates originates from the variation in metabolic burden imposed on each strain 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 burdened cells are quickly outcompeted by the more fit mutated cells which contain broken genetic circuits. They should be used with caution because they break almost immediately due to this stress. BBa_K3174004, containing a strong promoter and weak RBS, 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. 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.

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 isolates of each of the 5 parts with. The standard deviation for each bar 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 and contains a strong promoter. BBa_K3174006 and BBa_K3174007 have intermediate BFP expression levels because they are stable but contain weaker promoter/RBS combinations.

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]