Composite

Part:BBa_K3187015

Designed by: iGEM TU_Darmstadt 2019   Group: iGEM19_TU_Darmstadt   (2019-10-12)


Constitutively expressed chromoprotein amilGFP with Translation Enhancing 5'-UTR

Improved Version of BBa_K1073024

Usage and Biology

BBa_K3187015 (improved BBa_K1073024) encodes the yellow chromoprotein amilGFP derived from the coral Acropora millepora [5] under the control of a strong constitutive promoter (BBa_J23100) in combination with a ribosomal binding site ( BBa_B0032). The improved and the original version of the part were cloned into the pSB1C3 backbone and transformed in E. coli BL21 (DE3).

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.

Results



Fig. 2 shows the original version of the part (left) and the improved version (right) after overnight cultivation on an agar plate and after cultivation for 24 h in M9 minimal medium for better visualization of the emitted light at 512 nm [5] . Both the agar plate and the liquid culture were supplemented with chloramphenicol according to the cmp resistance on the pSB1C3 backbone. Both pictures were taken using a dark reader (Dark Reader Clare Chemical Research) for better visualization of the yellow color.



Figure 2: E. coli BL21 DE3 transformed with BBa_K1073024 after cultivation on LB-agar (left) and in a liquid culture (M9 minimal medium) supplemented with cmp. The improved version of the part shows increased amilGFP expression



Both the agar plate and the liquid culture show an enhanced expression of the amilGFP gene after the upstream insertion of the translation enhancing 5’-UTR.

References

  1. ↑ Peter O. Olins and Shaukat H. Rangwala, A Novel Sequence Element Derived from Bacteriophage T7 mRNA Acts as an Enhancer of Translation of the lacZ Gene in Escherichia coli, The Journal of Biological Chemistry, Vol. 264, No. 29, Issue of Ovtobet 15, pp. 16973-16976, 1989 [1]
  2. ↑ Shuntaor Takahashi, Hiroyuki Furusawa, Takuya Ueda and Yoshio Okahata, Translation Enhancer Improves the Ribosome Liberation from Translation Initiation, J. Am. Chem. Soc. 2013, 135 35, 13096-13106 [2]
  3. ↑ David K. Karig, Sukanya Iyer, Michael L. Simpson and Mitchel J. Doktycz, Expression optimization and synthetic gene networks in cell-free systems, Nucleic Acids Res. 2012 Apr; 40(8): 3763.3774 [3]
  4. ↑ Roberta Lentini, Silvia Perez Santero, Fabio Chizzolini, Dario Cecchi, Jason Fontana, Marta Marchioretto, Christina Del Bianco, Jessica L. Terrell, Amy C. Spencer, Laura Martini, Michele Forlin, Michael Assfalg, Mauro Dalla Serra, William E. Bentley and Sheref S. Mansy, Integrating artificial with natural cells to translate chemical messages that direct E. coli behavior, Nature Communications 5, Article number: 4012 (2014) [4]
  5. ↑ Joesfine Liljeruhm, Saskia K. Funk, Sandra Tietscher, Anders D. Edlund, Sabri Jamal, Pikkei Wistrand-Yuen, Karl Dyrhage, Arvid Gynna, Katarina Ivermark, Jessica Lövgren, Viktor Törnblom, Anders Virtanen, Erik R. Lundin, Erik Wistrand-Yuen and Anthony C. Forster, Enginerring a palette of eukaryotic chromoproteins for bacterial synthetic biology, Journal of Biological Engineering 12, Article number: 8 (2018) [5]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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