Preparation of competent E. coli by Inoue method
Competent cell refers to cells with capability of taking up extracellular genetic material. Chemically competent cells treated with buffer consisting calcium chloride (CaCl2) and other salts will disrupt the cell membrane, forming ‘holes’ allowing exogenous plasmids to enter the cell (Sigma, 2006). Competent cells are commonly produced chemically as they are less costly than electrocompetent cells. It is also a more convenient method for lab work as there is little requirement for special equipments. Inoue method is used in this lab which is a remarkable method as cells are cultured at temperature 18ºC instead of the conventional 37ºC. Typical transformation efficiency under standard laboratory condition is 1 x 108 to 3 x 108 transformed colonies/mg DNA plasmid. Bacterial cell culturing at 18ºC has shown best transformation efficacy. This condition might be due to cell membrane composition or physical characteristics of the cells synthesized at 18ºC, enabling better DNA uptake. The extension of growth cycle phases favoring efficient cell transformation is another hypothesis that might explain the condition (Joseph Sambrook, David W. Russell, 2012). Prokaryotic and eukaryotic cell competence is increased by dimethyl sulfoxide (DMSO) which penetrates living tissue without inflicting significant toxic damage on cells.
Ligation of DNA fragment into vector
Recombinant plasmid vector is produced by the insertion of novel gene into the plasmid, which is then used to transform cells. Ligation is the joining of two ends of DNA strands particularly3’hydroxyl of one nucleotide end and 5’phosphate of the other nucleotide end, corresponding to the cut end of the plasmid and the insert fragment (Colorado State University, 1999). Three current methods used for DNA ligation;
- Ligation of annealed cohesive ends produced by certain restriction enzymes using DNA ligase.
- Ligation of sticky or blunt ended fragments by T4 ligase from T4 phage-infected E.Coli.
- Synthesis of homopolymeric 3′ single-stranded tails at DNA fragment ends.
This lab utilizes T4 ligase which has the ability to ligate both sticky and blunt ended fragments. Ligation of blunt ended fragments is less efficient than sticky fragments due to absence of cohesive termini. Vector plasmid (puC18 or puC19) and DNA fragment are both restricted using similar restriction enzyme (EcoRI or BamHI) that recognizes specific sequences to produce compatible ends for ligation process. Ligation of novel DNA into MCS within lac Zgene of vector plasmid puC18/puC19 results in insertional inactivation of the gene thus inhibiting the production of lac Z gene product; which is then used for recombinant colonies screening. The plasmid vectors also carry ampicilin resistant gene, antibiotic resistance characteristic that will be acquired by successfully transformed cells (Introduction: Cloning (DNA Ligation) & Transformation). 2x ligation buffer is used to increase efficiency of DNA fragment ligation into plasmid vector. The buffer also functions to eliminate further purification before ligated DNA transformation (Promega Corporation, 2013). ATP supply is contained inside ligation buffer as energy source to T4 ligase activity (Ligation (Using T4 DNA Ligase), 2014).
Bacterial cloned plasmid transformation and blue/white screening
Transformation is a process where a cell ingests foreign DNA material from its external environment. The process happens readily in nature in some bacterial cells. Nowadays, scientists have found a technique to induce DNA uptake by bacterial cells in lab; by pore creation in bacterial cell membranes allowing DNA uptake. Heat shock method is used in the transformation of competent bacterial cells in this experiment where pre-cooled cell is heat shocked at 42C for an exact 45 seconds. The sudden temperature increment causes difference of pressure between inside and outside cell environment resulting in pore creation in cell plasma membrane. This enables DNA uptake into cell. Calcium chloride is utilized to produce environment rich in calcium which produces a counteraction toward electrostatic force between cell membrane of bacteria and plasmid DNA. Transformation efficiency can be calculated after transformed cell is plated. Successfully transformed bacteria are then used in cloning and amplification of desired gene (Journal of Visualized Experiments, 2014).
Bacterial spread on LBAIX plate consisting ampicilin allows survival of transformed cells containing plasmid with ampicilin resistance gene. Presence of X-gal in LBAIX plate produces blue and white colonies. White colonies are transformed bacterial cells with recombinant plasmid while blue colonies are transformed cells with non-recombinant plasmid vector. lacZ gene in the lac operon is utilized for blue white screening encodes for β-galactosidase protein that cleaves lactose into bromo-4-chloro-indoxyl, which undergoes spontaneous dimerization and oxidation into blue pigment 5,5′-dibromo-4,4′-dichloro-indigo. The color difference enables differentiation between transformed cells with and without recombinant gene. IPTG is an inducer that triggers binding of repressor protein to the inducer rather than the DNA molecule regulatory site, which usually keeps the operon ‘turned off’. This thus allows the transcription of lacZ gene which is then translated into protein enzyme that cleaves X-gal (EDVOTEK, 2005). The inactivation of β-galactosidase activity implies a successful vector recombination.
Bacterial plasmid isolation and restriction analysis
Plasmid is an accessory genetic element which virtually exists in all species of bacteria, yet with different copy number. Plasmid accounts a small portion of the whole bacterial genetic element with only approximately between 1 and 200 kb though exceptionally large plasmids have been found. Plasmid isolation procedure and solution used are based on the nature of plasmid which exists in supercoiled or also known as covalently closed circular (ccc configuration) configuration inside host cell (C. Rohde & B. Henze, 2011). The lab uses alkaline lysis method that utilizes 3 types of solution buffer containing principle solution SDS and highly alkaline sodium hydroxide base. Membrane phospholipid bilayer is denatured by detergent while the basic solution works to denature any protein involved in cell membrane structural maintenance (Plasmid Isolation (Mini prep), 2014). Several protocols are followed to eventually remove all cellular debris which leads to isolation and purification of plasmid. While other cellular structures are denatured by the series of steps in alkaline lysis, the supercoiled configuration of plasmid allows it to persist thus enables its isolation. Isolated plasmid DNA is then analyzed by using gel electrophoresis.
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Restriction endonuclease is an enzyme that cuts DNA at or near specific restriction sites. This enzyme is a vital tool in recombinant technology for the production of recombinant DNA (MOLE BIO/BIOCHEMISTRY, n.d.). Restriction enzyme used for the lab is EcoRI which forms sticky end with 5’ overhangs. EcoRI recognizes and cuts at GAATTC with rotational symmetry complementary sequence of CTTAAG. Plasmid cloning vector possesses a DNA sequence stretch that is recognized by specific restriction enzyme which is termed as polylinker or multiple cloning site (MCS). MCS is commonly located between promoter and terminator sequences, allowing cloning of DNA sequence by various restriction endonucleases (BioTechniques, 2001). In the lab, the product from restriction endonuclease digestion is analyzed by gel electrophoresis to detect the presence of insert DNA from the previous ligation method, thus implying the success of plasmid recombination.
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