Part:BBa_K2273033

Part Information
BioBrick Nr.	BBa_K2273033
RFC standard	RFC 25
Requirement	pSB1C3
Original Biobrick Part	BBa_K515005: sfGFP
sfGFP-His	BBa_K2273021: sfGFP
Submitted by	[http://2017.igem.org/Team:TU_Dresden TU Dresden]

Brief introduction in Fluorescent Proteins

 

Fluorescent proteins are small proteins with β-barrel-fold topology. They are useful for tracking global expression of target genes and localizations of these genes inside/outside cells. The unique chromophore in each fluorescent protein, originates from three intrinsic amino acids, at positions 65–67. The chromophore is tightly enclosed inside the protein and its formation does not require any cofactors or enzymes but only molecular oxygen. The rigidity of the β-barrel protects the chromophore from the environment and from radiationless decay. It also restricts chromophore flexibility as the correct folding of the protein is required for the chromophore formation. Proper orientation of the amino acids is necessary for chromophore maturation as it catalyzes chromophore synthesis.

Overview of sfGFP

 

A mutant of the wild-type green fluorescent protein from Aequorea victoria, called super folder GFP (sfGFP), is a new and robust derivation, designed for in vivo high performance analysis of protein expression levels. It demonstrates increased stability at higher temperatures and is able to tolerate protein tagging to poorly folding proteins while still maintaining fluorescence. It contains the important S65T mutation and F64L for red shift and folding respectively while it also has six additional mutations for enhanced folding: S30R, Y39N, N105T, Y145F, I171V and A206V. The absorption peak is at 480 nm while its emission peak is around 510 nm. (Cotlet et. al 2006)

sfGFP expression in Bacillus subtilis

For sfGFP, we used the Streptococcus pneumoniae codon adapted version, which was previously described to work best for B. subtilis (Overkamp et al. 2013). sfGFP was used to monitor the secretion of several different signal peptides and screen for best ones. To measure the contributions of sfGFP and native fluorescence at 511 nm, wild type and sfGFP-cloned B. subtilis were grown at density and then excited at 481 nm to obtain the emission peak amplitudes.

Figure 2: Fluorescence measured at 511 nm, excited at 481 nm, of sfGFP-cloned Bacillus subtilis and wild-type (WT) w168 strain, using a plate reader. The sfGFP gene was cloned in front of a Pveg promoter (BBa_K823003), a strong constitutive promoter in B. subtilis .

Secretion of sfGFP Analysis

Figure 2: Fluorescence fold increase of the supernatant, over the wild-type(W168 strain) supernatant, of various signal peptides secreting mCherry from Bacillus subtilis. The mCherry gene was cloned in front of a Pxyl promoter (BBa_K823015), an xylose-inducable promoter, and different signal peptides were used to test their ability to secrete mCherry.

Figure 2: Fluorescence fold increase of the supernatant, over the wild-type(W168 strain) supernatant, of various signal peptides secreting mCherry from Bacillus subtilis. The mCherry gene was cloned in front of a Pxyl promoter (BBa_K823015), an xylose-inducable promoter, and different signal peptides were used to test their ability to secrete mCherry.

References:

Overkamp, W. et al. Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl. Environ. Microbiol. 79, 6481–6490 (2013).



Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]