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miRNA-mediated regulation of synthetic gene circuits in the green alga Chlamydomonas reinhardtii Francisco Jose Navarro, and David C. Baulcombe ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00393 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019
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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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ACS Synthetic Biology
ACS Paragon Plus Environment
ACS Synthetic Biology Figure 2
site B site C
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ACS Paragon Plus Environment
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Figure 3
miRX mCherry
mVenus
miR1 miR2 miR3 miR4 miR5 miR6 miR7 n=71 n=62 n=82 n=73 n=69 n=72 n=73 n=79 n=80 n=79 n=80 n=69 n=76 n=19 n=42
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1 2 3 A 4 5 6 7 8 9 B 10 11 12 4 13 14 15 3 16 17 2 18 19 1 20 21 22 0 23 intronic 24 miRNA: 25 26 27 C 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
ACS Synthetic Biology
Name
Sequence
Repression activity
miR1 5’UAUGAAAGGCCUCUCACGCUG 3’ 82% miR2 5’UGACCGUUAUAGAAACGCCGC 3’ 93% miR3 5’UAAAGCCUAACGUUCGGCCGA 3’ 93% miR4 5’UCUAUACGUGGCAAAUCCCCC 3’ 69% miR5 5’UUUCAGCGAACGUAUACGCUC 3’ 67%
ACS Paragon Plus Environment3’ 66% miR6 5’UAUGUCUAGGCGAUCCCCCGU miR7 5’UAUAUCGGGACUCGGACCCCU 3’ 67%
ACS Synthetic Figure 4Biology
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1 2 3 mVenus 4 5.0 (+intronic miRNA) mVenus 5 4.0 6 mVenus mRNA 3.0 7 8 2.0 mCherry mRNA 9 1.0 10 RACK1 mRNA 11 0.0 12 2.0 miR1 13 14 miR1157 1.5 15 1.0 U6 16 17 1 2 3 4 5 6 7 8 0 1 9 10 11 0.5 18 19 0.0 1 2 3 4 5 6 7 8 0 1 9 10 11 20 mVenus mVenus 21 bp (+intronic miRNA) 500 22 300 100 23 24 2.0 25 mVenus mVenus 26 (+intronic miRNA) 1.5 27 28 7/7 1.0 29 5’UGCAGCGUGAGAGGCCUUUCAUACU 3’ mCherry mRNA 30 0.5 31 3’GUCGCACUCUCCGGAAAGUAU 5’ miR1 0.0 32 0 1 2 3 4 5 33 mVenus fluorescence (a.u.) 34 35 36 37 38mVenus (+intronic miRNA) 39 BF mVenus mCherry Merge 40 41 42 43 44 45 46mVenus BF mVenus mCherry Merge 47 48 49 50 ACS Paragon Plus Environment 51 52 53 54 55 56
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mVenus fluorescence (a.u.)
A
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mCherry fluorescence (a.u.)
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ACS Synthetic Biology
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