Difference between revisions of "Part:BBa K404002"

Latest revision as of 12:26, 31 October 2010

[AAV2]-Rep-VP123_p5-TATAless

Overview of RepVP123 plasmid

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 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 performed either by 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.

Figure 2 Table contains complete overview about all plasmids containing RepVP123 which were used by iGEM team Freiburg_Bioware 2010.

Modularization: Removing iGEM restriction sites and establishing loop insertion capability

Modifications in Rep

Making the RepVP123 wild-type compatible with the iGEM standards required the removal of five restriction sites (see Figure 1). This was achieved using site-directed mutagenesis for PstI (position 310) and PstI (4073). The remaining three iGEM restriction sites EcoRI (1578), PstI (1773) and EcoRI (1796) were replaced by a synthetic gene fragment, since the rep ORF contained these restriction sites in close proximity to each other plus an additional KpnI restriction site which was also not desired (see Figure 2). This gene fragment was cloned into the rep gene using HindIII and SwaI, which are single-cutting restriction enzymes adjacent to the target area. Additionally, BamHI (859) and SalI (1239) were removed, because these enzymes were required for genetically inserting the loop modifications in VP123.

Modifications in VP123

In order to implement the restriction sites necessary for targeting via loop insertions, the gene coding for the VP proteins was modified as well. The introduction of these restriction required up to four base mutations in a row, hence it was decided to synthesize this gene fragment and replace the wild-type sequence in RepVP123 as well.

Modularization: Adapting pSB1C3 to loop insertions – pSB1C3_001

To fulfill iGEM requirements all plasmids need to be submitted in pSB1C3, therefore primers were ordered for amplifying RepVP123 containing all modifications done so far by PCR and cloning the into pSB1C3. Still, pSB1C3 contains two restriction sites for SspI and PvuII restriction enzymes in its CAT marker. Since these are necessary for cloning ViralBricks in this vector, the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM Headquarters to implement a new standard for the pSB1C3 backbone which was named pSB1C3_001. Both restriction sites interfering with ViralBrick insertions were mutated to make SspI and PvuII single-cutters.

RepVP123 containing both rep and cap synthetic gene fragments including the re-mutation of KpnI and the downstream p5TATA-less promoter was cloned into the newly constructed pSB1C3_001. Testing this newly assembled plasmid in cell culture revealed unexpected data. Not only did the newly assembled plasmid work (see Figure 6), but in comparison to pAAV containing the same RepVP123 construct, pSB1C3_001 showed an about 3 times higher transduction efficiency. Although exact reasons are still unknown, these results are probably related to the reduced length of pSB1C3_001 compared to the original pAAV plasmid of approximately 1000 base pairs.

Sequence and Features