Table of Contents
Experimental design of plasmids helps in the understanding and application of genetic tools that are invariably important to our existence. All the natural organisms are born with specific immunity which is rare and unique to it's genomics, proteomics and metabol-omics. The genetic and technological manipulation provides a basis for transformation and if the transformation is successful,then there is commercialization of the product. Just like humulin developed by Eli Lilly to produce human insulin in E.Coli for mass production through bioreactor. The main objective of the experiment is to produce temperature and salinity resistance experimental design for ectoine expression in Synechococcus elongatus.
Fig.1 - Overall diagrammatic representation of plasmid construction starting from inserting of ect ABC to the final plasmid construct.
(Source: Author’s creation)
The plasmid construct is important to define how the foreign gene will be inserted and how it will function depending on its insertion into the plasmid. The plasmids used should have a high copy number and the product introduced should not cause intentional inactivation. This is an implied example of plasmid construct which is stunned up and transformed into the Synechococcus elongatus PCC 7942,a model organism. With high throughput technology the final plasmid is made. The subcloning is done to gain functionality and to study the desired gene in more detailed information and description. The figure represents how the plasmid vector is constructed. It is a small demonstration of theoretical framework in biotechnology.
1.Sequencing, analyzing ectoine gene cassette of the extremophilic bacteria Halomonas elongata from www.ncbi.nlm.nih.gov by using forward and reverse primers. The whole cassette of gene ectoine is selected.
2. The cassette of the gene is incorporated into pCR2.1 vector (which has f1 and pUC1 ori with T's site for cloning)-Invitrogen™
3.Vector system-pCR2.1 +entABC plasmid is designed
4. Sub Cloning is done with pSK+IBAT(so that functionality of ent ABC is shown under Pet.
5. Transformation is performed in Synechococcus elongate PCC-7942 se1Bb1s-eYFP followed by transformation procedure of Golden
Golden & Steams,1988)
6. Bioreactor cultivation (Growth and ectoine synthesis in recombinant S.elongatus)
7.Analysis-(Ono et al., 1999)
8.Protein Separation and identification Ectoine from the transformed cells
Experimental design- 3532bp fragment from the Halomonas was obtained with the help of its conserved sequences by performing SEFA PCR (SElf-Formed Adaptor PCR) and 2423bp entABC was selected along with 980bp sequence upstream and 129 sequences downstream and were cloned from Halomonas sp. with Genbank accession no: AJ011103. The sequences were identified by using reverse and forward primers. Ent a is around 579 bp, ent b is 1269bp and ent c is around 390bp. These genes are under the same transcription unit producing 21.2kDa, 49.4kd, and 14.7kd and these were the control of d0 and d1 promoters(Rouches & Lambert, 2020).
Experimental design- the vector pCR2.1 and the cloned sequence from Halomonas were inserted together then it was also subcloned with pSAK-IBAT which were experimentally designed by a group of scientists() and the desired site was introduced very carefully under Pet strong promoters and ent ABC has Xha1 and BamHI restriction endonuclease sites. So, the insert was under two origins of replication and three genes have the same transcriptional unit. The selectable markers used were ampicillin resistance and tetracycline resistance. The subcloning was done to get a good amount of ectoine protein depending on the regulation by Pet. The clone site of pCR2.1 was a TA site, initially, the ectoine gene was introduced there with pUC origin and PLac operon system (Zhang et al., 2020).
Experimental design- the Synechococcus were transformed with plasmid PASK-IBAT of 5200bp and were transformed according to Golden, 1987. The bacterium was grown in medium of (concentrations in mg liter1): (NH4)2SO4(6,000), K2HPO4(4,400);KH2PO4(3,400), CaCl26H2O (90), MgCl26H2O (1,100), glucose (10,000),ZnCl (3.5), MnCl2(0.46), CuCl2(7) and other sources of iron and molybdenum. And the transforming cells were selected by the presence of eYFP which acts as the selectable marker.
Bioreactor Cultivation, Analysis, and Protein Sequencing Were Done by Using the Known Protocols
Understanding the concept of ectoine pathway- Extremophlic bacteria like Halomonas elongata is an excellent example of osmoregular organism.it has cassette of gene which helps the organism to survey in the euryhaline condition of salt marshes and other brackish regions. These concepts intrigued the scientists and that started implying this technology in the plant biotechnology to make GMO crops especially in the crops that grow in extreme saline conditions such as the drought crops. The ectoine gene is a cluster of genes under the etc ABC and has a single transcription unit. Ent a codes for acetyl transferase, Ent b codes for amino transferase, and ent c codes for ectoine synthase. These are inserted under Ptet promoter.in our experiment and then it is introduced into Synechococcus elongatus. These bacterium is model transforming bacteria which was used by Golden in 1988 to simulate the utilization of the glucose in the bacteria.with the advancement and technological innovation there are more model species where this kind of experiments are performed to genetically modify our our crops to need the meeting demands of a growing population and it also helps in the protection and reviving of species in extreme conditions which has stenohaline tolerance. The methods and materials are explained as per the model species and its efficiency to transform and give higher amounts of ectoine which helps the organism to survive in various degrees of salinity. With these approaches we can also solve the problems of rising salinity in our soil with the increase in global warming.
This is a theoretical setup, which is designed based on experiments of different scientists all across the world. This construct might show a potential transformation in Synechococcus elongatus and in the similar way there can be chances of high infidelity in the plasmid expression vector. This experiment acknowledges all the important steps used for transforming a model bacterium.
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