Expression, Purification and Characterization of Novel Large Serine Integrase Assessment Answer

Abstract

With the advancement in the field of biotechnology at a rapid rate, the demand for biology is now more than ever. The use of highly efficient and sophisticated tools has been done for the precise manipulation of DNA. The bacteriophage integrase mainly catalyzes the recombination between an attP site, and this is done in circularized phage DNA. Serine integrase catalyzes the exact rearrangement of the DNA with the help of site-specific recombination of all the small sequences of DNA also known as attachment (att) sites. Unlike several other recombinases which are site-specific, the recombination reaction is mainly driven by high directional and it can also be reversed in the presence of some accessory protein also known as combinational directionality factor (RDF). Several legal and ethical considerations need to be followed and it is followed while conducting research. Well explained secondary research methodology has been followed and explained in the research. This study is highly significant as it promotes the integration which is site-specific of several mobile genetic elements in the bacterial component, hence, it should be promoted. Although there are several gaps in the research, hence, work should be work to address it.

Introduction

As the field of biotechnology is advancing at a rapid rate, the demand for biology is now more than ever. The use of highly efficient and sophisticated tools has been done for the precise manipulation of DNA. The bacteriophage integrase mainly catalyzes the recombination between an attP site, and this is done in circularized phage DNA. The attB site present in the chromosome of the bacterial host cell leads to the integrated prophage which is majorly flanked by attR and attL recombinant sites. Serine integrase belongs to the subfamily of serine site-specific recombinases, they are evolutionary and also distinct from the tyrosine site-specific recombinases (Olorunniji et al., 2017). Serine integrase catalyzes the precise rearrangement of the DNA with the help of recombination which is site-specific of all the small sequences of DNA also known as attachment (att) sites. Unlike several other recombinases which are site-specific, the recombination reaction is mainly driven by high directional and it can also be reversed in the presence of some accessory protein also known as combinational directionality factor (RDF). This ability to control the reaction in a directional form is the major reason for the development of serine integrase. The recombination sites which are present can be easily added towards the end of the DNA fragment, and the technology which is adopted is PCR technology with the help of primers. Several methods can be used for the expression, purification, and characterization of serine integrase (Durrant et al., 2022). In this research proposal, all these methods will be studied and it will be presented.

Objective

The objective of the research paper is to

1. Integrate gene and database
2. Integrase Gene into a vector and transport into E.coli
3. Expression and purification
4. Test the activity

Research Question

The research question is defined as:

The expression, purification, and characterization of a novel large serine integrase are studied, focusing on the bacteriophage ϕC31 and Bxb1 integrase and RDF protein.

Ethical Consideration

Several ethical considerations are followed when any gene is separated, and some of the ethical principles are affected such as autonomy, self-determination, and also self-governance. While performing any such research it is necessary to take care of all these points. Informed decisions need to be made and all practices need to be taken care of while performing any such activity. In this case, the right to autonomy also suggests the rights of the person to control the future use of the genetic material which is submitted for the analysis of some specific purpose. Although the principle of autonomy has been followed, some level of a breach might be experienced.

Some legal issues are also present, as people have the right to make an informed decision and follow the legal guidelines which are present. People have the right to be informed about and then the subsequent actions need to be taken. Some links need to be understood under federal regulations which govern the research involving human samples. In some cases conflict of interest might arise, hence, it should be noted that these conflicts of interest should be avoided. Confidentiality of all the experiments which have been performed should be studied and then actions should be taken directly. 

Research Methodology

Integrate Gene and Database

While collecting the data it is necessary to search for integrated genes and how databases are used to collect the information. The integrase gene is collected from the bacteriophage using several techniques, one such method which is used here is: the DNeasy Mini spin column is placed in a sterile microcentrifuge tube of 1.5 mL or 2 mL, and the pipette is used is 30 microliter. This buffer is directly poured into the DNeasy membrane. The tube is then incubated at room temperature for about 1 min and centrifuged for another minute at 6000g to elute DNA. The buffer solution and the protein purification process is a highly time-consuming process, a brief description is explained below (Dive & Newson, 2021). Pellet wash buffer should be prepared in the manner in which 20 mM Tris-HCl, pH is 7.5 and MgCl2 should be present in conc 10mM. Another component that is required is PMSF solution, buffer A, and buffer B is required and along with it recombinase dilution and dialysis buffer is required. This dilution buffer consists of 25 mM, pH 7.5, Tris HCl, 50% glycerol, and I mM DTT (Olorunniji et al., 2017).

Integrase Gene into a Vector and Transport into E.coli

The expression of plasmid in the E.coli strain BL21 pLysS with the help of calcium chloride transformation is done. Use of spread transformation reaction on the LB-agar plate is done to select pLysS and kanamycin for the pRT28-based expression plasmid, this is incubated overnight. As the cells are growing overnight, the pre-warm 400 mL 2X YT broth is incubated at 37℃. The culture then grows and it is shaken continuously and monitored every 20-30 min till the time it reaches 0.5-0.6 and this typically takes about 2-3 h. The culture is then cooled below 20°C at continuous shaking at 250 rpm (Cheng et al., 2021). Harvesting of cells is done and then it is centrifuged at 9800 g for 10 minutes at a temperature of 4°C. Some residual culture is obtained and then it is removed from the culture media or any soluble extracellular material, the pellets are then washed and again centrifuged at 9800 g for 10 minutes at a temperature of 4°C to re-pellet the cells. The cell pellets are weighed and then frozen at about -20 to -70°C to be used in the future (Chao et al., 2021).

Expression and Purification

The pellets are frozen and then thawed in the centrifuge tube at room temperature after that about 25 mL of the ice-cold buffer is thawed and it is scaled to suspend the cells in a smooth homogeneous paste with the absence of lumps. The cell suspension is obtained in a pre-cooled container so it can be used for sonication. This sample is then kept in ice, and three pulses are used for about the 20s at 30% amplitude and it is then cooled for 2 min in between each pulse. To this 100 mM phenyl methylsulfonyl fluoride (PMSF) is added to inhibit protease. The supernatant is then collected carefully and Ni2+ affinity column chromatography is performed to purify RDF and integrase protein (Makhija et al., 2018).

Test the Activity

The general applicability of the integrase-RDF fusion strategy is tested, and analogous methods are used to construct the fusion protein of Bx b1 integrase and the RDF gp47 (Ba et al., 2022).

Literature Review

Recombination with the help of large serine integrase is mainly used to integrate all the DNA fragments. When the in-built function of these proteins is studied it was studied that it is used to integrate the circular bacteriophage DNA in the host genomic DNA. The integrase also catalyzes the recombination between the attB site of host DNA and the attP site on the phage DNA. The integrated DNA is surrounded by attR and attL sites, and the recombination between the attR and attL requires the phage-encoded RDF along with integrase. Any DNA fragment which contains the attP site can easily join to the second DNA fragment with the attB site in the recombination reaction. The material which is used is ultrapure also known as double distilled water, which is used to form a solution for DNA biochemistry and protein purification. Deionized water which is used in the growth media and gel running buffer and bacteria media is purified for 20 min at 121 degrees Celsius (Merrick et al., 2018).

In another research paper by Olorunniji et al., (2017), it has been mentioned that serine integrase has been widely used for the application of molecular genetics, synthetic biology, and biotechnology due to its directionality, simple DNA sequence, and recombination efficiency. The plasmids which are involved in the in vitro analysis of integrase activity are studied. Other than this the two sites which are att sites are mainly created by implanting the synthetic double-standard att site oligonucleotide in pFM122. All the plasmids which are named are according to the att site and to describe vitro and in vivo sites are described. The plasmid in vitro is analyzed using the integrase activity, it also contains two att sites and implanting synthetic double-stranded att site oligonucleotide in pFM122. The plasmids are used for constitutive expression of integrase-RDF fusion and integrase protein in E-coli. The plasmids are used for the regular expression of the RDFs i.e. gp47 for Bxb1 integrase and gp3 for ϕC31 integrase. The ϕC31 integrase sequence which also includes His-tag C terminal was mainly derived from pARM010 along with the gp3 sequence and it was cloned from pEY301. The colon-optimized sequence of Bxb1 integrase and the RDF gp47 was retrieved from GeneArt. All the proteins which are extracted are extracted following the same protocols. These steps and protocols are highly useful and hence, they should be followed adequately. In vivo recombination reaction needs to be followed as it will help to modify the protein effectively and take adequate action. To have a fair idea about the integrase-mediated recombination in E-coli.

In another study by Li et al., (2018), it has been mentioned that the ability to modify, edit, and cloning of the DNA molecule will heavily rely on the bacteriophage enzyme which governs the phage-bacterial warfare. The manipulation of the eukaryotic genome and mainly the integration process is still a challenging process and it limits the growth in the field of synthetic biology. The phage and the bacterial integrase system is designed in a manner that is recombinase which is site-specific and along with it, it also inhibits some advantages. All these enzymes which have developed can catalyze the transfer of huge genetic payloads like the entire phage genome or the conjugative elements, collectively also known as mobile-genetic elements (MGEs). They are often tens of kilobase in length, and it shifts from one organism to another and it does not rely on the recipient's genetic repair machinery. The attachment sites need to be recognized and then the recognition sequence is found in the DNA donor along with the acceptor molecule. 

Significance of Study

This research is important because serine integrase is the DNA integrase that promotes the integration which is site-specific of several mobile genetic elements in the bacterial component. Serine integrase also catalyzes the excision and integration of the phage genome in and out of the bacterial chromosome. All of this is done in a highly directional and specific manner and it also makes the protein a highly powerful tool for genome engineering. In the year of 2013, the first structure of the serine-integrase DNA complex was developed and reported. In the serine recombinase activity, serine is required as it is a nucleophilic amino acid that is used by the enzyme to attack the DNA at the time of recombination which is site-specific. Other than this, in the case of tyrosine recombinase, tyrosine is a nucleophilic amino acid that is used by the enzyme to attack the DNA at recombination which is site-specific (Gupta et al., 2017). Serine integrase is also important as it helps to catalyze the insertion which is site-specific of the viral DNA in the host genome. It takes care of the precise requirements of DNA with the help of recombination which is site-specific of tiny sequences of DNA attachment. Other than this serine is also important as it is the key to the immune metabolite which modifies immunity as it controls the T cell proliferating capacity. If it is observed in terms of a mechanical manner, it can be easily determined that serine supplies one unit of carbon for the de novo biosynthesis of proliferating T cells. This research study is also important as it will help to mediate unidirectional recombination which is site-specific between the two DNA recognition sequences and the phage attachment site (Fogg et al., 2017).

Serine integrase can be widely used to recombine DNA, in both in vitro and in vivo wide spectrum, to record the presence of some biological signals which are used to carry the logic function. This in the future can also be applied in the self-programming and the decision-making biological machine. These machines can then be used in the disease diagnosis process, delivery and production of therapeutics, monitoring of remote environments, and bioremediation systems at the universal level (Li et al., 2020).

Gaps in the Research

Several gaps are present in the research and to obtain maximum productivity and result, it is necessary to address those gaps. The antibiotic stock solution should be diluted to 1000-folds and at a temperature of 55 ℃, the use of incubators is done, however, it should be avoided. The transformation which is done should be fresh and it should be done for every protein expression. If it is not fresh and not done for every protein then it will affect the result of the experiment. About 11 serine integrase have been identified which recombine with the att site of other integrase and present the result (Olorunniji et al., 2017). However, one major problem which is identified is that the current collection of tools related to serine integrase is very small. At maximum, there are only 14 biochemical serine integrase and 9 RDFs have been identified. If there are future advances made in the serine integrase technology it will help greatly in the evolution of some new strategies for the characterization and identification of these components. A greater understanding will be obtained of how to regulate and interact in recombination (Li et al., 2018).

References

Ba, F., Liu, Y., Liu, W. Q., Tian, X., & Li, J. (2022). SYMBIOSIS: Synthetic manipulable bio bricks via orthogonal serine integrase systems. Nucleic Acids Research , 50 (5), 2973-2985.

Chao, G., Travis, C., & Church, G. (2021). Measurement of large serine integrase enzymatic characteristics in HEK293 cells reveals variability and influence on downstream reporter expression. The FEBS Journal , 288 (22), 6410-6427.

Cheng, J., Ahmat, M., Guo, H., Wei, X., Zhang, L., Cheng, Q., & Zhang, R. (2021). Expression, purification, and characterization of a novel hybrid peptide CLP with excellent antibacterial activity. Molecules , 26 (23), 7142.

Dive, L., & Newson, A.J. (2021). Ethics of reproductive genetic carrier screening: From the clinic to the population. Public Health Ethics , 14 (2), 202-217.

Durrant, M. G., Fanton, A., Tycko, J., Hinks, M., Chandrasekaran, S.S., Perry, N.T., & Hsu, P. D. (2022). Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome. Nature Biotechnology , 1-12.

Fogg, P. C.M., Haley, J.A., Stark, W.M., & Smith, M. C.M. (2017). Genome integration and excision by a new streptomyces bacteriophage, ϕJoe. Applied and Environmental Microbiology , 83 (5), e02767-16. https://doi.org/10.1128/AEM.02767-16

Gupta, K., Sharp, R., Yuan, J.B., Li, H., & Van Duyne, G.D. (2017). Coiled-coil interactions mediate serine integrase directionality. Nucleic Acids Research , 45 (12), 7339–7353. https://doi.org/10.1093/nar/gkx474

Li, F., Dong, J., Lv, X., Wen, Y., & Chen, S. (2020). Recombinant expression and characterization of two glycoside hydrolases from extreme alkaliphilic bacterium Cellulomonas bogoriensis 69B4 ^T . AMB Express , 10 (1), 44. https://doi.org/10.1186/s13568-020-00979-8

Li, H., Sharp, R., Rutherford, K., Gupta, K., & Van Duyne, G. D. (2018). Serine integrase attP binding and specificity. Journal of Molecular Biology , 430 (21), 4401–4418. https://doi.org/10.1016/j.jmb.2018.09.007

Makhija, H., Roy, S., Hoon, S., Ghadessy, F. J., Wong, D., Jaiswal, R., & Dröge, P. (2018). A novel λ integrase-mediated seamless vector transgenesis platform for therapeutic protein expression. Nucleic Acids Research , 46 (16), e99-e99.

Merrick, C.A., Zhao, J., & Rosser, S.J. (2018). Serine integrases: Advancing synthetic biology. ACS Synthetic Biology , 7 (2), 299-310. https://pubs.acs.org/doi/abs/10.1021/acssynbio.7b00308

Olorunniji, F.J., McPherson, A.L., Rosser, S.J., Smith, M. C.M., Colloms, S.D., & Stark, W.M. (2017). Control of serine integrase recombination directionality by fusion with the directionality factor. Nucleic Acids Research , 45 (14), 8635–8645. https://doi.org/10.1093/nar/gkx567

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