Protein Engineers Must Know the Following Properties of Plasmids before Selecting Any Plasmid DNA as a Template

Importance of Plasmid DNA as a Template in Gene Technology

Protein Engineers must be acquainted with the basic and essential knowledge before going to use plasmid DNA as a template. Plasmid DNA as a template can play a significant role in Protein Engineering [1]. Plasmid DNA as a template is a good option to use in Protein Engineering because typically plasmids are beneficial for bacterial cells. In contrast, if viral DNA is used as a template, it can damage bacterial cells. 

1. Plasmid DNA is used as a template molecule for the insertion of the gene of interest. Inserted gene in the plasmid is exposed to oligonucleotides-directed mutagenesis using PCR as an amplification tool [1]. Later amplified plasmid along with inserted gene having incorporated the desired mutation is transformed into bacteria. 
2. Bacteria replication machinery replicates the plasmid and propagates during cell division to the daughter cells [2][3]. Along with plasmid replication, the transcription machinery of bacteria cells also transcribed the inserted genes along with other genes. 
3. These transcribed genes are translated into a protein product [4]. Desired synthesized protein is further evaluated for the introduced changes.

Keeping in view the above vital role of plasmid, selecting an appropriate plasmid DNA as a template can further enhance the chances of stably maintaining the plasmid DNA in the host cells as well as synthesizing in large quantities modified protein. The following facts will help us to select timely an appropriate plasmid DNA as a template.  

Note: Basic information about plasmid can be read in the following post, “Plasmid DNA mediated Oligonucleotide Directed Mutagenesis in Protein Engineering“

An Appropriate Copy Number of Plasmid DNA as a Template Can Increase the Amplification and Production of Gene of Interest

What is Copy Number?

Copy number can be simplified in such words that the normal number or average number of plasmids present in a signal bacterial cell [5]. Bacterial cells have two types of DNA molecules i.e., chromosomal DNA and plasmid. Irrespective of bacterial chromosomal DNA (i.e., present signal molecule/bacteria), the number of plasmid DNA is variable. Based on copy number, plasmids fall into two groups i.e., Stringent and Relaxed plasmids[6]. Both types can be differentiated from each other by specific properties.

1. Stringent plasmids remain attached to chromosomal DNA while relaxed plasmids remain free in the cytoplasm.
2. Due to the attached status, stringent plasmids replicate with chromosomal DNA. Whereas relaxed plasmids replicate independently of chromosomal DNA.
3. This is the reason that stringent plasmid copy number remains low and comparable with chromosomal DNA number i.e., approximately 1-5 copies /bacterial cell.
4. In contrast, relaxed plasmids increase their copy number during the life cycle of bacteria. Therefore, relaxed plasmids have high copy numbers like 10-50 copies /bacterial cell.

Based on copy number, using Relaxed Plasmids can help Protein Engineers to synthesize more products (i.e., a large number of DNA molecules) in the same host as compared to using stringent plasmid. Therefore, we suggest using Relaxed Plasmid DNA as a template for the synthesis of modified DNA products, having the desired mutation. While in case of modified protein expression low copy number plasmid is recommended [7] to synthesized modified protein, at commercial level. Low copy number plasmid has no burden of replication. 

Incompatibility between Plasmid DNA as a Template Can Severely Flop Our Designed Experimentations

Incompatibility (Inc) refers to the incapability of different plasmids to live together confidently in the same host at the same time [8]. Incompatibility is interrelated to the plasmids’ synchronicity in the host. Naturally bacteria comprise greater than one type of plasmid [9][10]. Diverse nature plasmids not only coexist stably in the bacterial cell but also remain unchanging in the subsequent generations. But occasionally two different nature plasmids decline living together stably in the host cell. Consequently, one plasmid loses as the division of host cells occurred. This miracle of Plasmids, negating stably coexisting in the host cell . Same members of the same Inc group, all such plasmids that cannot like co-existing in a stable manner in the same host at same times. However, If different plasmids exhibit coexisting stably, they are classified as members of the different Inc groups [11][12]. 

Being Protein Engineers, we must know about the plasmid(s) present in host cells and the nature of the plasmid that will be used for mutagenesis. In case any plasmid presents in bacteria and the plasmid DNA is used as a template both belong to the same Inc group [13]. In such a situation there is a chance that the plasmid DNA used as a template can be lost with the subsequent cell division of a bacterial cell. Therefore, plasmid DNA use as a template must be supported by host cells in presence of their own plasmids.

Host Range of Plasmid DNA Use as a Template Must be Broader.

Host range is one of the important features in selecting plasmid DNA as a template. Plasmids having a broad range of hosts, provide us an opportunity to replicate and express in multiple types of host cells [14]. We can opt for the best host easily in case of the broad range of plasmid. Broad host range plasmid will not bind us to one host.

Essential Components of Plasmid DNA Use as a Template in Protein Engineering

Structurally, Plasmids can be divided into three main components [15].

1. Origin of replication (ORI). Plasmids can maintain themselves in the host organisms by replicating themselves.]Origin of replication is the DNA sequence that allows initiation of replication of a plasmid. 
2. Selectable markers are genes that help us in a selection of bacteria colonies. Usually, plasmid having antibiotic genes facilitate bacteria to grow easily on antibiotic-added media. This is one of the useful features to choose the right clone of bacterial cells containing plasmid with our gene of interest. 
3. Multiple cloning site (MCS) consist of multiple restriction sites in a short segment of DNA allowing for the easy insertion of DNA. In the case of an expression vector using to express a gene of interest, MCS is often present downstream of a promoter. 

The above-mentioned points can significantly enhance the chance of getting great production by modifying our gene of interest through Protein Engineering tools mutagenesis experiments using an appropriate Plasmid DNA as a template.

References

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2. M. Shintani, Z. K. Sanchez, and K. Kimbara, “Genomics of microbial plasmids: classification and identification based on replication and transfer systems and host taxonomy,” Frontiers in Microbiology, vol. 6, Mar. 2015, doi: 10.3389/fmicb.2015.00242.
3. G. del Solar, R. Giraldo, M. J. Ruiz-Echevarría, M. Espinosa, and R. Díaz-Orejas, “Replication and Control of Circular Bacterial Plasmids,” Microbiology and Molecular Biology Reviews, vol. 62, no. 2, pp. 434–464, Jun. 1998, doi: 10.1128/mmbr.62.2.434-464.1998.
4. G. L. Rosano and E. A. Ceccarelli, “Recombinant protein expression in Escherichia coli: advances and challenges,” Frontiers in Microbiology, vol. 5, Apr. 2014, doi: 10.3389/fmicb.2014.00172.
5. M. Jahn, C. Vorpahl, T. Hübschmann, H. Harms, and S. Müller, “Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR,” Microbial Cell Factories, vol. 15, no. 1, Dec. 2016, doi: 10.1186/s12934-016-0610-8.
6. M. L. Kahn, D. Figurski, L. Ito, and D. R. Helinski, “Essential Regions for Replication of a Stringent and a Relaxed Plasmid in Escherichia coli,” Cold Spring Harbor Symposia on Quantitative Biology, vol. 43, no. 0, pp. 99–103, Jan. 1979, doi: 10.1101/sqb.1979.043.01.015.
7. S. L. Fontaine, S. D. Firth, P. J. Lockhart, J. A. Paynter, and J. F. B. Mercer, “Eukaryotic Expression Vectors That Replicate to Low Copy Number in Bacteria: Transient Expression of the Menkes Protein,” Plasmid, vol. 39, no. 3, pp. 245–251, May 1998, doi: 10.1006/plas.1997.1334.
8. Thomas, “Plasmid Incompatibility,” Springer New York, Jan. 01, 2014. https://link.springer.com/referenceworkentry/10.1007/978-1-4614-6436-5_565-2
9. N. S. Mitić, S. N. Malkov, J. J. Kovačević, G. M. Pavlović-Lažetić, and M. V. Beljanski, “Structural disorder of plasmid-encoded proteins in Bacteria and Archaea,” BMC Bioinformatics, vol. 19, no. 1, Apr. 2018, doi: 10.1186/s12859-018-2158-6.
10. D. Tomoiaga, J. Bubnell, L. Herndon, and P. Feinstein, “High rates of plasmid cotransformation in E. coli overturn the clonality myth and reveal colony development,” Cold Spring Harbor Laboratory, Mar. 2021. Accessed: Mar. 30, 2023. [Online]. Available: http://dx.doi.org/10.1101/2021.03.19.434223
11. W. C. Mutai, P. G. Waiyaki, S. Kariuki, and A. W. T. Muigai, “Plasmid profiling and incompatibility grouping of multidrug resistant Salmonella enterica serovar Typhi isolates in Nairobi, Kenya,” BMC Research Notes, vol. 12, no. 1, Jul. 2019, doi: 10.1186/s13104-019-4468-9.
12. M. Rozwandowicz et al., “Incompatibility and phylogenetic relationship of I-complex plasmids,” Plasmid, vol. 109, p. 102502, May 2020, doi: 10.1016/j.plasmid.2020.102502.
13. David P. Clark and N. J. Pazdernik, Molecular Biology. Elsevier, 2012, pp. 712–748.
14. A. Jain and P. Srivastava, “Broad host range plasmids,” FEMS Microbiology Letters, vol. 348, no. 2, pp. 87–96, Sep. 2013, doi: 10.1111/1574-6968.12241.
15. W. S. Klug, M. R. Cummings, C. A. Spencer, M. A. Palladino, and D. Killian, Concepts of Genetics. 2019, pp. 457–460.