Difference between revisions of "Part:BBa K2398009"

Revision as of 21:01, 1 November 2017

sd8-geneVI from M13 bacteriophage

This part provide bacteriophage M13 geneIII with a relatively weak RBS, sd8[1], flanked by two homology regions for the usage due to the cloning standard of the iGEM Team Heidelberg 2017 (http://2017.igem.org/Team:Heidelberg/RFC). Figure one gives a short overview of our standard. Our BioBricks from the registry can easily be used for the assembly of blasmid with the standard (Fig.: 2).

Figure 1: In our cloning standard, compatible building blocks are defined by specific functionalities. They are flanked by defined homology regions, indicated by numbers, which are necessary for the assembly of the APs with the Gibson method. This results in a highly customizable plasmid, composed of the desired origin of replication, an antibiotic resistance (4-5), a bicistronic operon with geneIII (2-3)and the desired reporter (3-4), which can be activated by any promoter (1-2)and a second expression cassette for additional genes that are necessary for the respective circuit (1-5).

Figure 2: Compatibility of our cloning stadard with the RFC10;Any AP building block can be cloned into RFC[10] standard by inserting BglII sites between the homology regions and the biobrick prefix or suffix, respectively. To use such a part for AP assembly, it has to be digested with BglII. The resulting fragment should be purified and can subsequently used for Gibson assembly with other parts.

This part was used in the context of a subproject of the iGEM Team Heidelberg 2017 (http://2017.igem.org/Team:Heidelberg/CRISPR).

Figure 3 gives a short overview on how the plasmid was used.

The AP consists of five subparts that are devided by homology regions for Gibson assembly (numbers). It carries an expression cassette for the transcription of a gRNA (between 1 and 5). GeneVI (2-4) is under control of a that can be activated by the Cas9-rpoZ in context with the respective gRNA. The whole plasmid can be produced with different origins of replication (4-5) to modulate the copy number and by exchanging the geneVI part with the RBS.

Sequence and Features

Rferences

[1] Ringquist, S.; Shinedling, S.; Barrick, D.; Green, L.; Binkley, J.; Stormo, G. D.; Gold, L. (1992): Translation initiation in Escherichia coli: sequences within the ribosome-binding site. In: Molecular microbiology 6 (9), S. 1219–1229.