Team:TU Darmstadt/Achievements/Improved parts
Part Improvement
Introduction
For the gold medal criteria Improved Part we improved the function of two different BioBricks (BBa_K1073024 and BBa_K1141000). The improved versions of the parts can be found here: BBa_K3187015 and BBa_K3187014. Both parts were improved in terms of their expression levels, 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 Takahashiet 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. Therefore, the 5'‑UTR 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: Improved Parts are showing increased Expression
Constitutively expressed chromoprotein amilGFP (BBa_K1073024)
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).
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.
Plac-RBS-mCherry-double terminator IPTG-inducible (BBa_K1141000)
BBa_K1141000 encodes the fluorescent protein mCherry under the control of an isopropyl-β-D-thiogalactopyranosid (IPTG)-inducible T7 promoter. Again, the translation enhancing 5'‑UTR was inserted upstream of the gene of interest (in this case mCherry BBa_J06504) for the improved version of the part. 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. 3) using a SpectraMax M5E.
Figure 3: 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.
Fig. 3 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. 4).
Figure 4: 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
- ↑ 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 E. coli, The Journal of Biological Chemistry, Vol. 264, No. 29, Issue of October 15, pp. 16973-16976, 1989 [1]
- ↑ 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]
- ↑ 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]
- ↑ 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]
- ↑ 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, Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology, Journal of Biological Engineering 12, Article number: 8 (2018) [5]
