Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon
Abstract
:1. Introduction
2. Results
2.1. Several Shortened Derivatives of the Shuttle Vector Are Stable in M. mazei
2.1.1. Elucidating Crucial Regions for Stable Replication in M. mazei
2.1.2. Smaller Shuttle Vectors Increase the Transformation Success
2.2. Smaller Shuttle Vectors in M. mazei Show the Same Phenotype and Copy Number as the Original pWM321
2.3. RepA Is the Essential Replication Protein
2.4. Interaction of RepA and DNA
2.5. RepA Binds and Nicks Archaeal Plasmids
3. Discussion
3.1. Optimisation of Archaeal Shuttle Vectors for Biotechnological Applications
3.2. RepA Shows Conserved Motifs of Rolling Circle Initiator Proteins
3.3. The Double-Strand and the Single-Strand Origin
4. Materials and Methods
4.1. Strains and Plasmids
4.2. Generation of Plasmids and Construction of Mutant Strains
4.3. Growth of M. mazei
4.4. Copy Number Determination
4.5. Purification of RepA-His6
4.6. Size-Exclusion Chromatography
4.7. Nickase Assay and Electrophoretic Mobility Shift Assay
4.8. Folding Transition Analysis Using Tycho NT.6
4.9. Pull-Down Analysis
4.10. Bioinformatic Alignment and Protein Structure Prediction
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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Name | Size (kbp) | Deleted Region | Area in pWM321 | Stability in M. mazei |
---|---|---|---|---|
pRS1532 | ~8.2 | ∆nc‘1 | ∆526–1287 | + |
pRS1515 | ~8.0 | ∆ssrA | ∆1316–2287 | + |
pRS1531 | ~8.4 | ∆nc‘‘1 | ∆2325–2778 | – |
pRS1518 | ~7.9 | ∆orf1+2 | ∆2811–3845 | – |
pRS1522 | ~7.2 | ∆repA | ∆3908–5688 | – |
pRS1523 | ~8.5 | ∆nc‘‘‘1 | ∆5712–6149 | + |
pRS1550 | ~6.5 | ∆nc‘ + ∆ssrA + parts of ∆nc‘‘1 ∆nc‘‘‘1 | ∆29–2556 ∆5712–6149 | + |
pRS1595 | ~5.8 | ∆nc‘ + ∆ssrA + parts of ∆nc‘‘ ∆nc‘‘‘1 | ∆29–2556 ∆5712–6149 | + |
Strain/Plasmid | Genotype/Relevant Characteristics | Source/Reference |
---|---|---|
M. mazei* | Potential cell wall mutant | [20] |
E. coli DH5α | General cloning strain | [43] |
E. coli JM109 λpir | General cloning strain | [43] |
E. coli BL21-CodonPlus®®-RIL | Overexpression strain with broader codon usage | Stratagene, La Jolla, CA, USA |
pWM321 | Shuttle vector E. coli–Methanosarcina (8.9 kbp) | [11] |
pET28a(+) | expression vector, N-term. His-tag | Novagene®®, Merck Millipore, Darmstadt, Germany |
pRS1452 | pWM321∆pac | This work |
pEX-K248_pac_JT | Synthesised pac gene under the control of pmcrB | Eurofins Genomics Life Science Services, Ebersberg, Germany |
pRS1515 | pRS1452∆ssrA+pac | This work |
pRS1518 | pRS1452∆orf1+2+pac | This work |
pRS1522 | pRS1452∆repA+pac | This work |
pRS1523 | pRS1452∆nc‘‘‘+pac 1 | This work |
pRS1531 | pRS1452∆nc‘‘+pac 1 | This work |
pRS1532 | pRS1452∆nc‘+pac 1 | This work |
pRS1550 | pRS1452∆29–2556, ∆5712–6149+pac | This work |
pRS1559 | pET28a(+)repA | This work |
pEX-K168_MCS_JT | Synthesised MCS | Eurofins Genomics Life Science Services, Ebersberg, Germany |
pEX-K248_pac2_JT | Synthesised pac gene under the control of pmcrB | Eurofins Genomics Life Science Services, Ebersberg, Germany |
pRS1595 | Modular shuttle vector | This work |
pRS1625 | pET28a(+)repA_DM | This work |
pBluescript II KS (+) | General cloning vector | Stratagene, La Jolla, CA, USA |
pRS1694 | pBSK with 345 bp region of pWM321 (ncts. 5088–5432) | This work |
Plasmid | Forward Primer | Reverse Primer | Purpose |
---|---|---|---|
pRS1452 | 5′-CGCCCGCCCCACGAC-3′ | 5′-CCTGCAGGTTTTGATGTAGTTTCTTACTAC-3′ | ∆6190–7150 from pWM321 |
pRS1532 | 5′-GATCCCGCAGATTATGGAAC-3′ | 5′-CAATTTCACACAGGAAACAGC-3′ | ∆526–1287 from pWM321 |
pRS1515 | 5′-GTATGTAAATAAATACTTTGTGC-3′ | 5′-GAAATAATGTTCCATAATCTGC-3′ | ∆1316–2287 from pWM321 |
pRS1531 | 5′-TTGTCGAAGAACTTCCAAAC-3′ | 5′-TAAATGACATCTATGCACAAAG-3′ | ∆2325–2778 from pWM321 |
pRS1518 | 5′-CGTATCACTTTAGGCTTTAAG-3′ | 5′-GATCGGTCTACTGTTTGGAAG-3′ | ∆2811–3845 from pWM321 |
pRS1522 | 5′-GAATAAGATTAACGCCTACC-3′ | 5′-CGTTCAACAAGGCTTTTG-3′ | ∆3908–5688 from pWM321 |
pRS1523 | 5′-CACTATCAAATGACATTGTAGTAAG-3′ | 5′-TAAGGTAGGCGTTAATCTTATTC-3′ | ∆5712–6149 from pWM321 |
pRS1550 | 5′-GTCACAACATTCACAAAAATAG-3′ 5′-CACTATCAAATGACATTGTAGTAAG-3′ | 5′-CCTGAATGGCGAATGGTTAAGG-3′ 5′-TAAGGTAGGCGTTAATCTTATTC-3′ | ∆29–2556 and ∆5712–6149 from pWM321 |
pRS1571 | 5′-GCGATCGCAACCTGCAGGTTCACTG-3′ | 5′-GATGTAGTTTCTTACTACAATGTC-3′ | Introduce AsiSI site in pRS1550 |
pRS1577 | 5′-GGCGCGCCTTAACTAGTCGCCATTCAG-3′ | 5′-GGTGGCACTGGCCGGCCAAATGTGCGCG-3′ | Introduce FseI/ SpeI site in pRS1550 |
pRS1559 | 5′-CGACAGGAAATGTCATATGAGTTCTGATTTTAG-3′ | 5′-GCCCAAAAGCCTTCTCGAGCGTGGCATCTC-3′ | RepA overexpression |
pRS1625 | 5′-GAGAGAAAAGATAAGTCACCTGC-3′ | 5′-CTTTTCTCTCACGTTGGGCAG-3′ | RepA _DM overexpression |
pRS1694 | 5′-GTTTTGGGCCCGGTTCGC-3′ | 5′-CTCCCGGGCCCAAGTCCATCGAAGC-3′ | Introduce putative DSO/ SSO in pBSK |
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Thomsen, J.; Schmitz, R.A. Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon. Int. J. Mol. Sci. 2022, 23, 11910. https://doi.org/10.3390/ijms231911910
Thomsen J, Schmitz RA. Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon. International Journal of Molecular Sciences. 2022; 23(19):11910. https://doi.org/10.3390/ijms231911910
Chicago/Turabian StyleThomsen, Johanna, and Ruth A. Schmitz. 2022. "Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon" International Journal of Molecular Sciences 23, no. 19: 11910. https://doi.org/10.3390/ijms231911910
APA StyleThomsen, J., & Schmitz, R. A. (2022). Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon. International Journal of Molecular Sciences, 23(19), 11910. https://doi.org/10.3390/ijms231911910