Difference between revisions of "Part:BBa K3971009"

Latest revision as of 18:21, 20 October 2021

Promoter characterization cassette for Synechococcus elongatus UTEX 2973.

This composite part was designed to characterize the strength of different promoters in S. elongatus UTEX 2973. The cassette contains two homology arms to the Neutral Site 1 of the S. elongatus UTEX 2973 genome which allows for the homology-repair-based insertion of the cassette into the genome. This cassette is to be cloned into the pSL2680 plasmid [1], which is a Cpf1 based CRISPR plasmid and can be used to insert the cassette into the Neutral Site 1 using a gRNA target within that region.

The promoter in this part is PcpcB-m6 (BBa_K3971000). In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 and BamH1 flank it, so that the promoter can be easily excised out and replaced with another and the cassette can be used to characterize that promoter.

Downstream of the promoter is a sYFP2 gene, and its fluorescence intensity can be used as a proxy for the strength of different promoters.

Both ends of the composite part contain Kpn1 restriction sites since this cassette is designed to be cloned into the pSL2680 plasmid (Addgene Plasmid #85581) which has a Kpn1 site and a Sal1 site to allow for cloning of cassettes.

Figure of the composite construct for promoter characterization. NS1a and NS1b are the two homology arms to Neutral Site 1.

References

1: Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria. Ungerer J, Pakrasi HB. Sci Rep. 2016 Dec 21;6:39681. doi: 10.1038/srep39681. 10.1038/srep39681 PubMed 28000776

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 2239
Illegal PstI site found at 362
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 2052
Illegal PstI site found at 362
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1103
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 2239
Illegal PstI site found at 362
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 2239
Illegal PstI site found at 362
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 152

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3971009 short</partinfo>
-This composite part was designed to characterize the strength of different promoters in S. elongatus UTEX 2973. The cassette contains two homology arms to the Neutral Site 1 of the S.elongatus UTEX 2973 genome which allows for the homology-repair-based insertion of the cassette into the genome. This cassette is to be cloned into the pSL2680 plasmid [1], which is a Cpf1 based CRISPR plasmid and can be used to insert the cassette into the Neutral Site 1 using a gRNA target within that region.
+This composite part was designed to characterize the strength of different promoters in <i>S. elongatus</i> UTEX 2973. The cassette contains two homology arms to the Neutral Site 1 of the <i>S. elongatus</i> UTEX 2973 genome which allows for the homology-repair-based insertion of the cassette into the genome. This cassette is to be cloned into the pSL2680 plasmid [1], which is a Cpf1 based CRISPR plasmid and can be used to insert the cassette into the Neutral Site 1 using a gRNA target within that region.
 The promoter in this part is PcpcB-m6 (BBa_K3971000). In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 and BamH1 flank it, so that the promoter can be easily excised out and replaced with another and the cassette can be used to characterize that promoter.
@@ Line 9: / Line 9: @@
 Downstream of the promoter is a sYFP2 gene, and its fluorescence intensity can be used as a proxy for the strength of different promoters.
-Both ends of the composite part contain Kpn1 restriction sites since this cassette is designed to be cloned into the pSL2680 plasmid which has a Kpn1 site and a Sal1 site to allow for cloning of cassettes.
+Both ends of the composite part contain Kpn1 restriction sites since this cassette is designed to be cloned into the pSL2680 plasmid (Addgene Plasmid #85581) which has a Kpn1 site and a Sal1 site to allow for cloning of cassettes.
 [[File:T--IISER-Pune-India--syfpcassette.png|thumb|700x900px|centre|Figure of the composite construct for promoter characterization. NS1a and NS1b are the two homology arms to Neutral Site 1.]]