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Plasmid cloning
Bacterial expression
Plasmid cloning with gene- plasmid having the gene of interest. Plasmid which is used is the T7 expression system. Following mRNA extraction from the tissue that has the highest concentration of the desired protein, reverse transcription is utilised to create cDNA from the various mRNAs. Open reading frame, or ORF, of the cDNA encoding the target protein is amplified by PCR using gene-specific primers, and at the same time, extra sequences are introduced that contain restriction sites for cloning the DNA into a vector [1]. Thermal Cycler (PCR machine) was used to amplify the cDNA. In one cycle of PCR, the amount of cDNA gets doubled
Plasmid transformation
The cloned plasmid was introduced into bacteria. Incubation of plasmid with bacteria as well as using the materials given below by carefully placing the cells in them:
Equipment required
Heat block (set at 42֯ C) duration of 60sec thermal current produced drawing plasmid into the cell.
Equipment use
The heat block is majorly utilized for inducing transformation shock at specific temperatures and thus the sample is heated at a certain temperature. The heat block makes the cells capable of foreign DNA entry into the bacterial cells. Recovery Broth which can be LB broth is slowly added to the cell suspension as well as plasmid bacterial cells are allowed to recover for a period of thirty minutes at a temperature of 37°C. The device has high precision as well as convenience [2], the instrument is quite suitable for getting accurate results as well as temperature control required for transformation.
Capability
The recovery phase in the heat block allows the bacteria for effectively repairing their cell walls for effective gene expression. It is capable of ensuring a safe as well as clean, work environment due to its proven ability to provide superior heating properties.
Incubation phase
It is known as the recovery phase as well as incubation was done for a period of approximately 1 hour at 37֯ C (180-200rpm)
Equipment use
The incubator shaker is utilized for the growth of cells as well as bacterial along with yeast cells.
Equipment capability
The incubator Shaker has the capability of maintaining the temperature and other environmental conditions for the transformed samples. It allows setting up a range from compact as well as bench-top systems to certain large-capacity [3]. Furthermore, this shaker series helps in allowing time for bacterial cells to production of the antibiotic-resistant gene.
colony selection
- The agar plate will be placed in the facility as well as left in the incubator. Selection of colony will be done followed by pre-culturing with antibiotic (small amount 10-50ml) Step 5: Inoculation: Incubated overnight at 37֯ C (180-200rpm) in a shaking incubator. Then inoculation of the culture (larger flask 500ml-3L) will take a few hours.
Growth phase
The mid-log growth phase (for bacteria) is reached by using a particular piece of equipment.
Equipment use
Spectrophotometer is utilized as an analytical instrument used which has the objective calculation of visible light as well as UV light followed by infrared light emission or reflection. The Spectrophotometers are helpful in measuring the intensity in relation to the function of the wavelength of a source of light [4].
Equipment capability
The capability of the spectrophotometer is to measure the optical density of the sample via measuring light intensity lying in the whole electromagnetic spectrum, the range of spectrometers involves ultraviolet as well as visible followed by infrared light and near-infrared ranges.
Then induction phase using IPTG which helps the bacteria express the protein.
Lysis phase
Equipment use
The equipment is C5 Emulsiflex which is helpful in the lysis phase at the lab scale as well as majorly air driven at high-pressure homogeniser which quickly decreases particles as well as droplets from the size of micron to nanometer.
Equipment capability
The capability of the C5 Emulsiflex is its high-end use in lysis. This equipment is capable of high-pressure homogenisation along with use in the lysis of emulsion along with liposome processing. The Emulsiflex C5 utilizes a dynamic homogenising valve [5]. The advantage involves that it has a dynamic valve that helps in processing samples at uniform homogenising pressure. It helps in the production of very uniform liposomes along with microemulsions which have particle size distribution which is quite narrow.
Centrifugation step
Equipment use
Centrifugation was done by using Multifuge X3R Centrifuge (short) at 4֯C for 10-30mins 4000xg (low temperature- protects the protein). Then pellet will be produced in form of membranes with protein
The multitude refrigerated x3r centrifuge is used for separating materials having different densities as well as particle sizes in liquid via generating relative centrifugal force [6] for the plasmid cell sample.
Equipment capability
It has a maximum speed of 15200 rotations per minute as well as a maximum relative centrifugal force [7]. It has high capacity due to varying rotors involving fixed Angle rotors as well as swing-Out rotors which improve its RCF as well as capacity.
References
- Langlais C, Korn B. Recombinant protein expression in bacteria. Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine, Springer, Berlin Heidelberg. 2006:1609-16.
- Konczal J, Gray CH. Streamlining workflow and automation to accelerate laboratory scale protein production. Protein Expression and Purification. 2017 May 1;133:160-9.
- Hausjell J, Kutscha R, Gesson JD, Reinisch D, Spadiut O. The effects of lactose induction on a plasmid-free E. coli T7 expression system. Bioengineering. 2020 Jan 6;7(1):8.
- Greenwich JL, Alakavuklar MA, Fuqua C. An Inducible T7 Polymerase System for High-Level Protein Expression in Diverse Gram-Negative Bacteria. Microbiology Resource Announcements. 2023 Jan 16:e01119-22.
- Arai T, Aikawa S, Sudesh K, Kondo T, Kosugi A. Electrotransformation of thermophilic bacterium Caldimonas manganoxidans. Journal of Microbiological Methods. 2022 Jan 1;192:106375.
- Green MR, Sambrook J. Cloning and transformation with plasmid vectors. Cold Spring Harbor Protocols. 2021 Nov 1;2021(11):pdb-top101170.
- Green MR, Sambrook J. The Inoue method for preparation and transformation of competent Escherichia coli:“Ultracompetent” cells. Cold Spring Harbor Protocols. 2020 Jun 1;2020(6):pdb-rot101196.
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