Part:BBa_K1141000

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:

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

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Improved version: BBa_K3187014

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 and Takahashi et al. . 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. .

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)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. a spacer between the epsilon motive and the RBS improves the translation rate.
GAAGGAG	According to Karig et al. and Lentini et. al a distance of 4-9 bases between RBS and start codon increases the translation efficiency.
AATAATCT	According to Lentini et. al an AT-rich composition between the RBS and the start codon results in the best expression results.

Results

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Both the original and the improved version were cloned in pSB1C3. E. coli BL21 (DE3) was transformed using BBa_K1073024 and cultured using 25 mL LB medium supplemented with Chloramphenicol (based on the pSB1C3 resistance). The expression was measured (Ex: 587 nm, Em: 610 nm) over a period of 6 hours after induction with 0.5 mM IPTG (Fig. 2) using a SpectraMax M5E.

Figure 2: Expression of mCherry with (improved BBa_K1141000) and without (BBa_K1141000) the translation enhancing 5’ untranslated region was measured (Ex: 587 nm, Em: 610 nm) over a period of 6 hours after induction with 0.5 mM IPTG using a SpectraMax M5E.

Figure 2 shows an increased expression of the improved BBa_K1141001 in comparison to BBa_K1141001. The increased expression rate of the improved version (left) after 6 hours can also be observed with the naked eye (Fig. 3).

Figure 3: E. coli BL21 (DE3) cultures transformed with BBa_K1141000 (right) and the improved version of BBa_ K1141000 (left) after cultivation in LB medium supplemented with Chloramphenicol (according to the resistance provided by pSB1C3) for 6 hours.

References