Cloning of SOD Gene into E. coli Using pUC19 Vector

Categories: Biology

Essay, Pages 10 (2257 words)

Views

Abstract

Transformation is a process of uptake of DNA by competent bacteria. Escherichia coli (E. coli) is one of the bacteria that is not naturally competent and need to be induced. Artificial transformation such as calcium chloride method can be used to prepare a competent E. coli. A cloning vector that commonly used for the transformation of E. coli is pUC19. Vector pUC19 is a circular double stranded plasmid with 2686 base pairs that has high copy number. The foreign gene that will be inserted into pUC19 is superoxide dismutase (SOD) gene.

Don't use plagiarized sources. Get your custom paper on

“ Cloning of SOD Gene into E. coli Using pUC19 Vector ”

Get high-quality paper

NEW! smart matching with writer

SOD gene code for an enzyme called superoxide dismutase that can break down toxic to avoid damage of cells. After the SOD gene is inserted into the pUC19, the plating of competent E. coli was performed to identify the colony that successful carrying a plasmid with SOD gene in it. The transformed clone were verified by using bacterial colony PCR.

Introduction

Polymerase chain reaction (PCR) is a technique used to amplify or copy small specific segments of DNA.

One of the uses of PCR is, it can be used in forensic field. PCR can be used in genetic fingerprinting as a tool. This technique can be used to identify a person from millions based on DNA profile (Hugget & O’Grady, 2014). For example, they also write that DNA or a DNA database of suspects can be compared with samples of DNA that has been collected from crime scene. This technique can identify or rule out suspects during a police investigation.

There are several steps that involved during PCR cycles. The first step is denaturing stage where the double stranded DNA template is heated at 94 – 95 ºC to break the hydrogen bond in order to separate them into a single strand.

Next, is annealing stage where the DNA strand is cooled down to 50 - 65 ºC to enable the primer to attach to DNA template. The primer served as starting point for DNA synthesis and it is designed to be complementary to the short sequence of DNA on each end of the sequence to be copied. The last stage is extending stage. At this final step, the heat is increased to 72 ºC to allow synthesize of new DNA made by Taq DNA polymerase enzyme which add DNA bases.

DNA purity and concentration are important to be measured because the measurement of DNA concentration might be inaccurate due to DNA impurity and it could potentially affect subsequent labelling reaction (Oxford Gene Technology, 2011). DNA concentration is measured to know the quantity of DNA while DNA purity is measured to determine the quality of DNA. In measuring DNA concentration, using spectrophotometer is better than agarose gel because it gives more accurate and quantitative results. Moreover, the gel electrophoresis takes more time to run.

Agarose gel electrophoresis separates DNA fragments based on the size. This method is usually use to view the DNA fragment that have been digested with restriction enzyme (Khalsa, 2010). She also states that this method is frequently used in forensic technique and DNA fingerprinting Gel electrophoresis is a method to isolate DNA fragment based on their size and charge which involve running a current through the medium in that contain molecules of interest (Khan Academy, n.d.). The phosphates of DNA molecules is negative, therefore the DNA fragments will migrate to positive pole (Bhatia & Dahiya, 2015)

DNA transformation is a process where foreign DNA is inserted into a host cell (Addgene, 2017). On agar plates, satellite colonies formation is caused by the degradation of ampicillin. Satellite colonies are small colonies that does not take up the plasmid that form around a large colony that contain antibiotic resistant gene (Oswald, n.d.). The number of transformed cells generated by 1 µg of supercoiled plasmid DNA in a transformation reaction is called transformation efficiency. The transformation efficiency can be calculated as follow:

Transformation efficiency = x 1000 ng / µg

Bacteria contain operon which is a functioning unit of genomic DNA that is controlled by a single promoter that contains a group of gene (Reina, 2017). Lac operon is normally switch off and will turn on when the glucose level is low and there is a presence of lactose. Khan Academy (n.d.) writes that lactose act as inducer and will be broken down to produce allolactose. The allolactose will bind to lac repressor which will cause conformational changes to the lac repressor and making it cannot bind to operator (Khan Academy, n.d.). Thus, the lac operon is turned on and can be transcribed by RNA polymerase.

Materials and Method

Preparation of Competent Escherichia coli Cells

In order for Escherichia coli to do transformation, the cells must be competent to take up foreign DNA into the cells. E. coli cells can become competent either through natural transformation of artificial transformation. Natural transformation is when bacteria take up the foreign DNA or plasmid naturally. Artificial transformation is the bacteria need to be induced by chemical or electroporation in order to become competent to take up the foreign DNA. Naked DNA can be introduced into the bacteria by using chemical or electroporation method to make the bacteria become competent. By using chemical and electroporation method the bacteria can become artificially competent.

Electroporation is a method where electric current is passed through the cell wall. It also written that the current can create momentary “pores” in cells membranes and the negatively charged DNA will be forced into cells (E. coli electroporation vs chemical transformation, n.d.). Electroporation must be performed at 0ºC to minimize heat damage to the cells. E. coli become chemically competent after being resuspended into CaCl2 solution at 0ºC. Ca2+ ion creates pores in membrane and ease the passage of DNA through hydrophobic cell membrane (E. coli electroporation vs chemical transformation, n.d.). It also written that DNA is force into cells by applying short heat shock.

Transformation technique is useful in genetic engineering because it is one of the ways to create recombinant DNA. It is also can be used to make DNA cloning and to make large scale of specific human proteins. Transformation can be done naturally or artificially on competent cells. During experiment, glycerol was added to chemically competent E. coli in order to protect the cells from crystallization when stored under liquid nitrogen. The cells are grown until the OD550 is between 0.3 – 0.5 because the cells is at growing phase where the cells grow fast and healthy. If the cells grown at 0.8 of OD550, the cells will enter stationary phase which is not suitable for transformation and they also will less survive the freezing process. E. coli cells were incubated at 37ºC in LB broth which is also known as Luria-Bertani broth which is a broth that allow rapid growth of E. coli.

Plasmid Extraction and Isolation

PCR machine was programmed to undergo 29 cycles because for amplification of the DNA. The process took 2 hours long and be able to produce multiple the DNA that was desired to yield. Patel (2009) states that the purpose final extension part is to promote complete synthesis of PCR products by allowing the reannealing the PCR product into double stranded DNA so it can be used for visualization by using ethidium bromide after gel electrophoresis of for cloning. Different DNA gene cannot use same primers. For example, SOD primer can only be used to amplify SOD genes while bla primer can be used to amplify bla genes. This is because the primer is designed to be complementary to the short DNA sequence and different DNA gene have different DNA sequence.

PCR products are purified and analysed by running on agarose gel electrophoresis by laboratory demonstrators. There was a band detected and from a sample while the other one has no band detected (Group 12). The one with detected band shows that the SOD gene was cleaved by the restriction enzymes. The sample with no band indicate that the SOD gene was not cleaved by the restriction enzymes. This results could be due to the error occur during the collection of content at the bottom of Eppendorf tube after microcentrifugation or the pellet were resuspended too harsh. Since only few groups have the SOD gene digested, it was divided equally to all group for the use in the next practical.

Table 1: Absorbance Readings for Spectrophotometer and NanoDropTM

Equipment	OD at 230 nm	OD at 260 nm	OD at 280 nm
Spectrophotometer	0.067	0.071	0.032
NanoDropTM	0.111	0.091	0.055

Calculation

DNA concentration:

DNA concentration is calculated by using this formula:

DNA concentration (μg/ml) = OD260 x 50 x dilution factor

Spectrophotometer: DNA concentration = 0.071 x 50 x20 = 71 μg/ml

NanoDropTM = 4.3 μg/ml (The concentration of DNA was displayed on the machine)

Calculation of DNA purity:

Spectrophotometer:

A260 /A280 = 0.071 / 0.032 = 2.219

A230 /A260 = 0.067/ 0.071 = 0.944

NanoDropTM.

A260 /A280 = 0.091 / 0.055 = 1.655

A230 /A260 = 0.111/ 0.091 = 1.220

Spectrophotometry can be used to determine the purity of DNA solution by comparing optical density at different wavelength. For pure DNA, the observed 260/280 nm ratio will be near 1.8 and elevated ratios will usually indicate the presence of RNA. While, ratios below 1.8 indicate the presence of contaminating protein or phenol. Based on the calculation, the 260/280 value for spectrophotometer is 2.219 and for NanoDropTM is 1.655. The result shows that, when using spectrophotometer, there is a presence of RNA while for NanoDropTM, the result indicates that there is a presence of contaminating protein or phenol. To further support the presence of contamination of protein or phenol, 230/260 ratios can be calculated. If the ratios is greater than 0.5, there is a protein or phenol contamination.

Based on the calculation, the 230/260 ratios for spectrophotometer is 0.944 and for NanoDropTM, the value is 1.220. This shows that the DNA is contaminated with protein and phenol as the ratios is greater than 5. Based on this practical, the best method to determine the purity of DNA is by using NanoDropTM. This is because it is more accurate than spectrophotometer and using cuvette can lead to many errors. For example, the cuvette can be dirty or have scratch and the reading can be inaccurate. To eliminate RNA from DNA, RNase A can be used to remove RNA contamination (Geneaid, n.d.). Phenol- chloroform extraction can be used to remove protein from DNA sample (New England Biolabs, 2007). The reading of absorbance can be improved by making sure the cuvettes is cleaned before used and the take three readings of absorbance to obtain average reading.

Both A and B undigested plasmid were represent by the thickest band. The undigested plasmid produce three band because it consist of supercoiled and open circular undigested plasmid with extra twist and it also has more intense dye because it holds the dye better (SCL, n.d.). The digested plasmid can be seen as a single band on 6th lane. Both HindIII and BamhII is a type II restriction enzyme which cut at specific site. Both of the enzyme cut at the specific restriction site in SOD gene and pUC19. Plasmid size is usually reduced when it comes to construct vector from naturally occurring plasmid because it can produce high copy number.

DNA is negatively charged because it contain backbone phosphate. The purpose of adding dye to the DNA sample before loading it into the wells of agarose gel is because to give a visualization to naked eye (Chin, n.d.). RNase was added into DNA to degrade RNA which can affect the DNA impurity. RNase- untreated plasmid can be detected by using electrophoresis where the RNA will be migrating ahead of DNA (Oxford Gene Technology, 2011).

Next, for the plates containing ampicillin and LB with sample from tube C shows abundant growth. However, this plate should not contained any growth as the sample is consist of competent cell that does not contain plasmid with ampicillin resistance gene. Next, the agar plates containing LB, ampicillin, X-gal and IPTG for sample A and B contained both white and blue colonies.

Transformation efficiency = x 1000 ng / µg

Based on Table 1 the both plate of sample A and B that contain LB, ampicillin, X-gal and IPTG have two distinct colony consist of blue and white colonies. Blue-white screening is used to identify recombinant bacteria. The non-recombinant bacteria form blue colonies while the recombinant bacteria will form white colonies (Aitken, 2012). The white colonies indicate the lacZ gene is disrupted and replaced by SOD gene. The blue colonies the absent of SOD gene. There is no growth in the -pUC bacteria spread in the nutrient agar supplement with ampicillin because there is no plasmid, therefore ampicillin resistance gene is absent. The antibiotic cannot be degraded as the ampicillin resistance gene is absent. Thus, the bacteria will be killed by the antibiotic.

During each of the inoculation, it is important to re-flame the spreader to sterilise the spreader to avoid any contamination. The plates that contain only LB form lawn on the agar because there is no antibiotic is present while the plates with antibiotic on the agar will form as colonies instead of lawn. Satellite colonies formed around large blue colonies that have bla-containing plasmid does not contain pUC19 (Oswald, n.d.). He also states that the satellites colony form because the blue colonies released beta-lactamase that degrades the ampicillin.

If the LB plate contain kanamycin instead of ampicillin the satellite colony will not form as the blue colonies contain only ampicillin resistance gene not kanamycin resistance gene. The blue colonies will also not be formed as the kanamycin kill the bacteria. X-Gal is used for the screening of clones containing recombinant DNA. β-galactosidase will hydrolyse X-gal into galactose and an insoluble pigment, 4-chloro-3-brom-indigo (Welch, 2015). The non-recombinant bacteria will take up the blue dye and form blue colonies. IPTG is an analog of galactose that induces the expression of lacZ gene (Sigma Aldrich, n.d.).