Difference between revisions of "Part:BBa K3165044"

Latest revision as of 01:54, 21 October 2019

RBS + i^2mCherry (Leu)

This part consists of an improved version of mCherry (BBa_J18932) that reduces the truncation caused by the wild type protein with a ribosome binding site upstream of the coding sequence.

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
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 718
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

I² mCherry (Leu) is an improved version of mCherry (BBa_J18932), which is widely used as a fluorescent marker. However, N-terminal fusion of proteins with mCherry is not suited for studying various signal peptides due to the significant truncation that arises due to the presence of an RBS like sequence upstream of the ninth amino acid, Met which happens to be coded by the start codon (ATG). The RBS like sequence along with the start codon causes the transcription to begin at an internal site, resulting in significant truncation during protein expression. In order to completely shut down the truncation caused by mCherry, we modified the internal start codon (ATG) via 2 single base mutations at the 46th and 48th nucleotides to convert ATG to CTT (coding for Leu). In the absence of a start codon, no transcription is expected, thus eliminating the chances of any truncated products.
Along with the RBS sequence, the i²mCherry (Leu) forms a functional translational unit by providing a binding site for the ribosome.

This part can be used under a suitable promoter and terminator as a generator for i²mCherry (Leu), for use as a fluorescent marker.

@@ Line 11: / Line 11: @@
 ===Usage and Biology===
-i2mCherry (Leu) is an improved version of mCherry(BBa_J19832), which is widely used as a fluorescent marker. However, N-terminal fusion of proteins with mCherry is not suited for studying various signal peptides due to the significant truncation that arises due to the presence of an RBS like sequence upstream of the ninth amino acid, Met which happens to be coded by the start codon (ATG). The RBS like sequence along with the start codon causes the transcription to begin at an internal site, resulting in significant truncation during protein expression.
+I<html><sup>2</sup></html> mCherry (Leu) is an improved version of mCherry <html><a  href="https://parts.igem.org/Part:BBa_J18932">(BBa_J18932)</a></html>, which is widely used as a fluorescent marker. However, N-terminal fusion of proteins with mCherry is not suited for studying various signal peptides due to the significant truncation that arises due to the presence of an RBS like sequence upstream of the ninth amino acid, Met which happens to be coded by the start codon (ATG). The RBS like sequence along with the start codon causes the transcription to begin at an internal site, resulting in significant truncation during protein expression.
 In order to completely shut down the truncation caused by mCherry, we modified the internal start codon (ATG) via 2 single base mutations at the 46th and 48th nucleotides to convert ATG to CTT (coding for Leu). In the absence of a start codon, no transcription is expected, thus eliminating the chances of any truncated products.<br>
 Along with the RBS sequence, the i<sup>2</sup>mCherry (Leu) forms a functional translational unit by providing a binding site for the ribosome. <br>