Difference between revisions of "Part:BBa K2273033"

Revision as of 09:12, 22 October 2017

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 (GFP) from Aequorea victoria, super folder GFP (sfGFP). sfGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. sfGFP shows increased thermal stability and is able to tolerate genetic fusion to poorly folding proteins while remaining fluorescent. It incorporates the red shift S65T mutation and the folding mutation F64L and six additional mutations which improve its folding: S30R, Y39N, N105T, Y145F, I171V and A206V. (Cotlet et. al 2006)

Modularization: Overview

In our terminology the term “RepVP123” encompasses the whole AAV2 genome excluding the ITRs. The rep locus comprises four proteins related to genome replication while the cap locus codes for the proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which are required for viral capsid assembly. Source of the RepVP123 BioBrick supplied within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the wild-type AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In order to introduce the iGEM standard and additionally enabling the possibility to modify the viral capsid via integration of certain motives within the viral loops 453 and 587, a total of twelve mutations within RepVP123 (see Figure 1) and additionally two mutations within the pSB1C3 backbone were introduced by either Site-Directed Mutagenesis (SDM) or by ordering and cloning of specifically designed gene sequences matching the required demands. Modifying the pSB1C3 led to iGEM team Freiburg_Bioware’s variant of this backbone, pSB1C3_001.