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Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
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2018 (English)In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 12, article id 8Article in journal (Refereed) Published
Abstract [en]

Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.

Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.

Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.

Place, publisher, year, edition, pages
BIOMED CENTRAL LTD , 2018. Vol. 12, article id 8
Keywords [en]
Chromoprotein, Fluorescent protein, Coral, Escherichia coli, Genetic marker, Reporter gene, Integration, Fitness cost, BioBrick, iGEM
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-356454DOI: 10.1186/s13036-018-0100-0ISI: 000432246200001PubMedID: 29760772OAI: oai:DiVA.org:uu-356454DiVA, id: diva2:1235928
Funder
VINNOVASwedish Research Council, 349-2006-267Swedish Research Council, 2011-5787Swedish Research Council, 2016-1Swedish Research Council, 2017-04148Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceAvailable from: 2018-07-30 Created: 2018-07-30 Last updated: 2019-01-25Bibliographically approved
In thesis
1. Exotic Ribosomal Enzymology
Open this publication in new window or tab >>Exotic Ribosomal Enzymology
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis clarifies intriguing enzymology of the ribosome, the multiRNA/multiprotein complex that catalyzes protein synthesis (translation). The large ribosomal RNAs (23S and 16S rRNAs in E. coli) are post-transcriptionally modified by many specific modification enzymes, yet the functions of the modifications remain enigmatic. A deeper insight into two of the 23S rRNA S-adenosyl-methionine-requiring methyltransferase enzymes, RlmM and RlmJ, was given by investigating substrate specificity in vitro. Both enzymes were able to methylate in vitro-transcribed, modification-free, protein-free, 2659-nucleotide-long 23S rRNA. Furthermore, RlmM was able to methylate the 611-nucleotide-long Domain V of the 23S rRNA alone and RlmJ could modify the A2030 with only 25 surrounding nucleotides.

Translation is evolutionary optimized to incorporate L-amino acids to the exclusion of D-amino acids in the cell. To understand how, and how to engineer around this restriction for pharmacological applications, detailed kinetics of ribosomal dipeptide formation with D- versus L-phenylalanine-tRNA were determined. This was done by varying the concentrations of EF-Tu (which delivers aminoacyl-tRNAs to the ribosome) and the ribosome, as well as changing the tRNA adaptor. Binding to EF-Tu was shown to be rate limiting for D-Phe-tRNA at a low concentration of EF-Tu. Surprisingly, at a higher (physiological) concentration of EF-Tu, binding and subsequent dipeptide synthesis became so efficient that D-Phe incorporation became competitive with L-Phe, and accommodation/peptide bond formation was unmasked as a new rate-limiting step. This highlighted the importance of D-aminoacyl-tRNA deacylase in restricting translation with D-amino acids in vivo.

Although polypeptides are intrinsically colorless, it is remarkable that evolution has nevertheless enabled ribosomes to synthesize highly colored proteins (chromoproteins). Such eukaryotic proteins reside in coral reefs and undergo self-catalyzed, intramolecular, chromophore formation by reacting with oxygen in a manner highly similar to that of green fluorescent protein. The potential utility of different colored chromoproteins in E. coli was analyzed via codon-optimized over-expression and quantification of maturation times, color intensities and cellular fitness costs. No chromoprotein was found to have the combined characteristics of fast maturation, intense color and low fitness cost. However, semi-rational mutagenesis created different colored variants with identical fitness costs suitable for competition assays and teaching.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 43
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1770
Keywords
rRNA modification, Methyltransferase, RlmM, RlmJ, D-amino acid, Unnatural amino acid, Chromoprotein
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-374965 (URN)978-91-513-0567-7 (ISBN)
Public defence
2019-03-08, A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-02-15 Created: 2019-01-25 Last updated: 2019-03-18

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