Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reporter Modification
2.2. Thiolation Domain Insertion and Co-Expression
3. Results
3.1. Construct Generation and Domain Insertion
3.2. Indigoidine Production
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primers | |||
---|---|---|---|
Primer Name | Sequence 5′:3′ | Length | Annealing °C |
BpsA_outF2 | TCCAGCACCTGATGATGAAC | 20 | 58.4 |
BpsA_outR2 | CTGGATGCCGTAGAACGAG | 19 | 59.5 |
BpsAhindiiiR1 | GACGCCAAGCTTCGCGTTGAGCTCGCGGACGAGGCCGACGGCGATCAGCGA | 51 | 91.1 |
BpsAhindiiiF1 | CAACGCGAAGCTTGGCGTCTCCCTGCCGCTGCAGAGCGTCCTGGAGTCC | 49 | 89.6 |
BpsAafliiR1 | CTCGCGCTTAAGGGCCTTCTCCCAGACCGCCGCGATCTCCTTCTCCGT | 48 | 88.5 |
BpsAafliiF1 | AGAAGGCCCTTAAGCGCGAGAACGCCTCCGTCCAGGACGACTTCTTCG | 48 | 86.4 |
Inserts | |||
Insert Name | Sequence 5′:3′ | Binding Site Amino Acid | |
3KS_1 | GAATCGGGCATGGACTCAAAAGCAGCCCTTGTTCTG | ESGMDSKAALVL | |
3KS_2 | GAATTGGGCTTAGATTCTTTGTCCGGCGTTGAATTT | ELGLDSLSGVEF | |
3KS_3 | GAAAGCGGAATTGATTCCTTGTCTGCAGTAGAGTTT | ESGIDSLSAVEF | |
3KS_4 | GAGAGTGGCATGGACTCATTATCTGCCGTCGAGTTT | ESGMDSLSAVEF | |
BurA_1 | GCT TCA GGT GCA GAA TCT ATC GCT GTC GTG GGC GTG | ASGAESIAVVGV | |
BurA_2 | CAA TTA GGA TTA GAC AGC TTG GAA ACC GTT CAA CTG | QLGLDSLETVQL | |
ZmaK_1 | GAA ATC GGT GGG CAC TCG CTG TTA GCA ATG AAA CTT | EIGGHSLLAMKL | |
ZmaK_2 | GAT GCC GGG TTA GAT AGC TTA TCC TTA ATT AGC TTA | DAGLDSLSLISL | |
5′ Linker † | AGAAGGCCCTTAAGCGCGAGAACGCCTCCGTCCAGGACGACTTCTTC | ||
3′ Linker † | GTCCGCGAGCTCAACGCGAAGCTTGGCGTCTCCCTGCCGCTG |
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Williams, E.; Bachvaroff, T.; Place, A. Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter. Microorganisms 2022, 10, 687. https://doi.org/10.3390/microorganisms10040687
Williams E, Bachvaroff T, Place A. Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter. Microorganisms. 2022; 10(4):687. https://doi.org/10.3390/microorganisms10040687
Chicago/Turabian StyleWilliams, Ernest, Tsvetan Bachvaroff, and Allen Place. 2022. "Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter" Microorganisms 10, no. 4: 687. https://doi.org/10.3390/microorganisms10040687