Reporter

Part:BBa_K1141000

Designed by: Adrien Rapeaux   Group: iGEM13_Grenoble-EMSE-LSU   (2013-09-23)
Revision as of 22:20, 18 October 2019 by PeterGockel (Talk | contribs)

Plac-RBS-mCherry-double terminator (IPTG-inducible)

This part contains all the necessary DNA to produce the red fluorescent protein mCherry with the PLac promoter. PLac repression is low in most E. coli strains and the promoter will be leaky. Efficient repression can be implemented by additionally transforming a strain with the pREP4 plasmid from the M15 E. coli strain of QIAGEN, or transforming the part into this strain. The figure below shows the excitation and emission spectra as can be obtained from CHROMA's spectra viewer:

Excitation-Emission spectra for mCherry

Figure 1: Excitation (left peak) and Emission (right peak) spectra for mCherry.

Translation Enhancing 5'-UTR (Improvement by iGEM TU_Darmstadt 2019)

Usage and Biology

Improved version: BBa_K1758100

The part was improved in terms of its expression level, based on the insertion of a 5’-untranslated region (5’-UTR) upstream of the coding sequence. This 5'-UTR was adapted from iGEM Bielefeld 2015 (  BBa_K1758100) and is based on the research of Olins et al [1] and Takahashi et al. [2] . It contains the strong ribosomal binding site (RBS) g10-L from the T7 bacteriophage and a sequence that plays a role in the regulation of mRNA binding to and release from the 30S ribosomal subunit. The 5'-UTR therefore enhances the translation efficiency of the following coding sequence (CDS) (Fig. 1).

Figure 1: Schematic depiction of the composition and interaction of the enhancer sequence with the 30S ribosomal subunit described by Takahashi et. al. [2] .

The sequence of the translation enhancing 5’-UTR can be divided into the four main features listed below:



Sequence Function
AATAATTTTGTT
TTAACTTTAA 		The T7 g10 leader sequence (first described by Olins et al [1] )increases the efficiency of translation initiation. This sequence contains the epsilon motif TTAACTTTA which enhances the binding of the mRNA to the 16S rRNA.
poly-A Referring to Takahashi et al. [2] a spacer between the epsilon motive and the RBS improves the translation rate.
GAAGGAG According to Karig et al. [3] and Lentini et. al [4] a distance of 4-9 bases between RBS and start codon increases the translation efficiency.
AATAATCT According to Lentini et. al [4] an AT-rich composition between the RBS and the start codon results in the best expression results.
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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None