The process of making a synthetic cell takes three stages i.e. design, synthesis, assembly and transplantation of base pairs that can give a DNA code with well defined functions. The first process (design process) involves mapping of the natural DNA from an organism whose DNA is to be artificially synthesized. This leads to the development of a genome sequence that is digitized. In Venter’s case Mycoplasma mycoides was synthesized by using its digitized sequence that was sequenced and its essential genes identified and mapped out. The mapping of Mycoplasma mycoides forms part of the design stage. After a complete sequence is mapped and developed non-essential parts of the sequence are avoided during the synthesis process as long as they do not affect the replication or survival process of the organism and new sequences acting as “water marks” are inserted into the synthesized DNA sequence pieces in order to identify the new DNA pieces (Venter et al. 2010).
The DNA pieces created in the synthesis process are then assembled using the right media and later selectively transplanted into living cells such as bacteria, whereupon; they take up their own life and begin self replication, taking on a life of their own which is independent of the host cell’s DNA control. These new cells have phenotypic properties that can be defined and predicted from the design stage which involved mapping of the DNA of the natural cells from which their sequence was obtained. The placement of part of Mycoplasma mycoides DNA sequence in to the Mycoplasma capricolum cell takes over the control of the new cell and initiates protein synthesis that are characteristic of the synthetic DNA sequence inserted into the cell, thus giving it properties that are different from its natural form-hence the synthetic connotation.
Transformansts screening
After completing the process of DNA transfers into other cells, whether artificial or not there is a need to identify the number of cells that have undergone a transformation in a colony that was undergoing DNA introduction. The screening process that identifies bacterial transformants from the process described above needs to employ a mechanism that can enable sorting of the transformants from the non-transformants. Screening for these recombinant plasmids within transformants is enabled through the use of vectors which have detectable genes that can be detected visually. These genes are known as reporter genes and they code for the production of some colored proteins that are used in blue/white colony identification processes. Phenotypic alterations on colony in these systems occur when exogenous DNA interrupts a gene (vector borne indicator gene). E.Coli’s LacZ system is commonly used for this end.
The principle of white and blue selection of positive recombinants is simply a mode of identifying successful recombination in the transfer process. Bacterial LacZ gene on plasmids produces (b-gal) b-galactosiadase, and this occurs if the ORF encounters no interruption from the DNA which is inserted. The non-transformants in this case are expected to have a blue color that results from the alpha peptide that results from hosting e.coli, whose introduced plasmid produces b-galactosiadase, which in turn causes the blue color in X-gal. However, if a foreign gene had been introduced (MCS) multiple cloning site, there would be changes on the LacZ gene which will impede production of b-galactosidase and therefore, the colony remains white, rather than blue (Venter et al 2010).
DNA methylation
DNA methylation is a process via which the fifth position in a pyrimidine cytosine ring is added with a methyl group or alternatively the nitrogen on the sixth position of the purine adenine rings gets methylated. This process of modification is inherited as cells divide. The process of methylation is extensively studied in bacteria, and it is cited as process through which DNA arrangement and replication occurs. The methylation process is catalyzed by various (DNA-MTasas) DNA methyltransferases. The mechanism of methylation serves a defensive purpose in bacteria. This protects a bacterial cell from being affected by foreign DNA introduced into its system. Endonucleases are responsible for this restriction, and as such they are able to discriminate between foreign and endogenous DNA, thus preventing its influence after identifying its methylation pattern. Newly introduced DNA that is not methylation protected is cleaved and eliminated.
Methylation also controls fidelity of the replication process. The identification of the methylation process and its control is important in the process of introducing new DNA because it determines whether the new DNA will be rejected by the cell expected to host it for a transformation or not. Therefore, in the DNA transfer process keen care and attention has to be dedicated to the process of methylation. The process is also maneuvered to cleave DNA and allow insertion of genes (Rubens & Chaffin, 1998).
Genetic watermarks
The genetic water marks denote areas of insertion of new genetic material that serves to differentiate the new synthesized genes from the natural gene sequence of their original mapping. These water marked portions have a characteristic identity of inserted genes that are supposed to express a certain phenotype when they manifest. This acts as an identity and serves to show the expression of the newly inserted material. The watermarked zones are areas that contain genes of least significance to the mapped natural genome in terms of replication and multiplication and as such they cannot. The differentiation property accorded enables the scientists to identify the transformants and differentiate them from natural cells. Some of the replaced portions may contain restriction endonucleases and these are later used to identify patterns in the transformants through selective digestion (Venter et al 2010).
The future of synthetic cell making
Craig Venter believes that synthetic cell technology is the future key, because it can allow for control over genetic manipulations with greater precision of mapping with very little to no guess work. The mapping of genomes of various organisms as expressed by Venter can help identify important phenotypic traits that can be applied in development of new organisms that can help in solving a multitude of environmental problems that exist at the moment. Venter also claims that their approach is far more precise because it combines a multitude of processes such as multiple deletions, insertions and substitutions all of which help to shape the gene mapping in a manner that earlier methods would have not been able to do (Venter et al (2010). The method provides the possibility to do genomic mapping and make computer generated desired genetic and phenotypic types in a very simple manner compared to other earlier methods which lacked the ability to make such maneuvers with genes in organisms. DNA sequencing that is computer enabled allows the storage of genomic material even in electronic files with clear and easy mapping which may extend to eternity, and thus; Venter praises it as the future of genomics.
References
Venter et al (2010), Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome; Science Journal: volume number 329, issue number 5987 pp 52-56
Rubens, C. E. and Chaffin, D. O. (1998),. Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression, retrieved on 6th April, 2011 from http://www.ncbi.nlm.nih.gov/pubmed/9757005
