Difference between revisions of "Part:BBa K3165013"

Revision as of 19:33, 20 October 2019

i^2mCherry (Ile)

An improved alternative of mCherry (BBa_J18932) designed to minimise the amount of truncated protein by modifying the internal start codon of the existing 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 679
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

Biology

i²mCherry (Ile) 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.
The 2018 IIsc-Bangalore team attempted to reduce the truncation of the mCherry sequence by modifying the internal RBS like sequence and decreasing its ability to pair with the ribosome. Their improved part imCherry (BBa_K2609006) reported a considerable decrease in the truncation but they couldn't get rid of the truncated product entirely.
In order to completely shut down the truncation caused by mCherry, we modified the internal start codon (ATG) via a single base mutation at then 48th nucleotide to convert ATG to ATC (coding for Ile). In the absence of a start codon, no transcription is expected, thus eliminating the chances of any truncated products.

Usage

i²mCherry(Ile) was developed to overcome the shortcomings of the existing mCherry (BBa_J18932) BioBrick which is not suitable for protein fusion studies due to the truncation faced at the N-terminal. As a consequence of reduced truncation, i²mCherry can be used for studying signal peptides and other N-terminal protein fusion components.

Characterization

Expression with BBa_K3165046

The protein was expressed under the T7 promoter in Escherichia coli (BL21DE3) with 6xHistag at the N-terminal. The transformed bacteria were incubated at 37^oC for over 4 hours. The cells were lysed by sonication and the lysate was collected via centrifugation. The lysate was run on an SDS PAGE which showed two bands. The size of the protein of interest along with the 6xHistag is around 27 kDa. The upper band corresponds to the non-truncated protein while the lower band represents the truncated product.

References :

(1) https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609006

@@ Line 21: / Line 21: @@
 i<sup>2</sup>mCherry(Ile) was developed to overcome the shortcomings of the existing mCherry (BBa_J18932) BioBrick which is not suitable for protein fusion studies due to the truncation faced at the N-terminal. As a consequence of reduced truncation, i<sup>2</sup>mCherry can be used for studying signal peptides and other N-terminal protein fusion components.
+<h2> Characterization </h2>
+<html>
+<h3> Expression with <a href="https://parts.igem.org/Part:BBa_K3165046">BBa_K3165046</a></h3></html>
+The protein was expressed under the T7 promoter in <i>Escherichia coli</i> (BL21DE3) with 6xHistag at the N-terminal. The transformed bacteria were incubated at 37<sup>o</sup>C for over 4 hours. The cells were lysed by sonication and the lysate was collected via centrifugation. The lysate was run on an SDS PAGE which showed two bands. The size of the protein of interest along with the 6xHistag is around 27 kDa. The upper band corresponds to the non-truncated protein while the lower band represents the truncated product.
 <h3> References : </h3>