application of biochemistry methods in e coli through transformation plasmid purification pcr and gel electrophoresis

In this study, various molecular biology techniques were utilized with Escherichia coli JM109 and the plasmid pBR322.. This technique relies on exposing the bacteria to a sudden increase

International University, Vietnam National University – HCMC

SCHOOL OF BIOTECHNOLOGY DEPARTMENT OF APPLIED CHEMISTRY

REPORT

PROJECT

Application of biochemistry methods in E.Coli through transformation, plasmid purification, PCR, and gel

electrophoresis

Instructor: MSc Le Tran Hong Ngoc

Teaching Assistant: Ms Trinh Thi Xuan

Group: 02

Group members:

Date of submission: June 28th

, 2024

I ABSTRACT

DNA, a molecule within cells, carries the genetic blueprint essential for an organism's growth and functionality Plasmids, distinct from chromosomal DNA, are small, circular DNA molecules capable of autonomous replication In this study, various molecular biology techniques were utilized with Escherichia coli JM109 and the plasmid pBR322 The competent JM109 strain of E coli, chosen for its ability to uptake foreign DNA, was prepared for transformation using a heat-shock method This technique relies on exposing the bacteria to a sudden increase in temperature, which creates pores in the bacterial cell membrane through which the plasmid DNA can enter The plasmid pBR322, containing genes for ampicillin and tetracycline resistance, was used in the transformation experiment Despite controlled conditions, no colonies were observed on the agar plates, indicating a failure in the transformation process, with contamination noted in the initial set of plates The presence of gram-negative E coli was confirmed through Gram staining, a method that distinguishes bacterial species based on the structural differences in their cell walls The successful preparation of plasmids using the QIAprep Spin Miniprep Kit, which employs alkaline lysis followed by silica membrane-based purification, yielded purified plasmid DNA for PCR amplification However, the results of gel electrophoresis, a technique used to separate DNA fragments by size, indicated no visible bands, suggesting that the PCR amplification did not produce the expected DNA fragments This study highlights the importance of optimizing experimental conditions for the successful transformation and amplification of plasmid DNA

in molecular biology research

DNA is a molecule found inside cells that carries the genetic information necessary for an organism's development and function The term 'plasmid' refers to a DNA molecule that is distinct from chromosomal DNA and can replicate independently These small, circular DNA molecules are known for their ability to replicate autonomously, as they do not depend on the organism's chromosomal DNA for this process A pure DNA which was essential for protein synthesis and protein quantitation In this study, Escherichia coli JM109 and the plasmid pBR322 were selected for use in several techniques including transformation, gram staining, plasmid preparation, PCR, and DNA electrophoresis The choice of these organisms and [1]

techniques was based on both theoretical and practical considerations

Gram staining is a fundamental microbiological technique used to differentiate bacteria based

on their cell wall composition Gram-positive bacteria stain purple due to their thick [3]

peptidoglycan layer, while gram-negative bacteria stain pink because of their thinner peptidoglycan and outer membrane The process involves applying crystal violet, iodine, a decolorizer, and safranin[1] In this study, gram staining was employed to verify the purity of the E coli JM109 strain and detect contamination in other samples, highlighting its importance

in bacterial identification and analysis

A cell undergoes transformation when its DNA is updated by information from its surroundings Although rare, this procedure could happen naturally in some species of bacteria Bacteria could be made to take in DNA from their surroundings under specific laboratory circumstances "competent" bacteria are those that can quickly absorb DNA from their surroundings Making cells competent increased the DNA permeability of their cell membrane The cell uses the new DNA after it has entered the bacteria to produce RNA and subsequently protein The new proteins that this DNA generates are what alter the characteristics of the cells This experiment was carried out using plasmid pBR 322 Two genes that code for resistance [1]

to antibiotics are present in it; one of them codes for resistance to ampicillin, and the other for resistance to tetracycline Bacteria are typically chosen as the transformation host because they have developed the capacity to take in foreign DNA and duplicate it quickly to obtain vast amounts From a theoretical standpoint, E coli is a well-established model organism commonly used in molecular biology research The competent JM109 strain of E coli employed in the experiment allowed it to take up DNA that otherwise would not pass through the membrane Furthermore, JM109 has a mutation in the lacI gene, which reduces the likelihood of false positives in the screening process

Heat-shock transformation is a pivotal technique in molecular biology, extensively used to introduce foreign plasmid DNA into bacterial cells, most notably Escherichia coli This [2]

method plays a crucial role in genetic engineering, cloning, and various biotechnological applications The process involves several key steps, including the preparation of competent cells, the addition of DNA to the cells, the heat shock treatment, and the subsequent cultivation

of the transformed bacteria to calculate the transformation efficiency The process begins [2]

with the preparation of competent bacterial cells, which are treated with a solution of calcium

chloride This treatment increases the permeability of the bacterial cell membrane, making it more susceptible to DNA uptake The cells are then mixed with the plasmid DNA and subjected

to a brief heat shock, typically at 42°C for 30-60 seconds This sudden increase in temperature induces the formation of transient pores in the cell membrane, through which the plasmid DNA can enter the cells Following the heat shock, the cells are rapidly cooled on ice to close the pores and stabilize the membrane The transformed cells are then incubated in a nutrient-rich medium to allow them to recover and express the genes carried by the plasmid DNA This method is highly efficient and widely used due to its simplicity and effectiveness, enabling researchers to manipulate bacterial genomes, produce recombinant proteins, and conduct various forms of genetic analysis and experimentation

Plasmid preparation is a technique for extracting and purifying plasmid DNA It was a crucial [4]

step in many molecular biology investigations and necessary for the effective application of plasmids in biotechnology and research Plasmid DNA from bacteria had been purified using

a variety of techniques The plasmid DNA is frequently isolated from contaminating proteins and genomic DNA during the purification process Small-scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way to perform clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion) Lysing, binding, washing, and elution are the four phases, and spin column-based nucleic acid purification is used to complete each stage Plasmid DNA purification was [4]

carried out in this task using the QIAprep Spin Miniprep Kit, 33% faster than usual

Figure 1 Plasmid preparation process for the QIAprep Spin Miniprep Kit [4]

Polymerase chain reaction (PCR) is a common laboratory technique used to make copies (millions or billions) of a particular region of DNA Typically, the goal of PCR is to make [5]

enough of the target DNA region that it can be analyzed or used in some other way For

instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis[5] PCR is based on the ability of DNA polymerase to synthesize new strands

of DNA complementary to the provided template strand The DNA template is the sample DNA that contains the target sequence At the beginning of the reaction, a high temperature is applied

to the original double-stranded DNA molecule to separate the strands from each other DNA polymerase a type of enzyme that synthesizes new strands of DNA complementary to the is target sequence The first and most commonly used of these enzymes is TaqDNA polymerase Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR: they can generate new strands of DNA using a DNA template and primers, and they are heat resistant Because DNA polymerase can only add a nucleotide to a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide Primers are short pieces

of single-stranded DNA that are complementary to the target sequence The polymerase begins synthesizing new DNA at the end of the primer Nucleotides (dNTPs or deoxynucleotide triphosphates) are single units of the bases A, T, G, and C, which are essential "building blocks" for new DNA strands At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons) PCR is used in many areas of biology and [5]

medicine, including molecular biology research, medical diagnostics, and even some branches

of ecology In this study, PCR was used to amplify the DNA fragment of interest from the purified pBR322 plasmid The amplified DNA fragment was then further analyzed and [1]

cloned into other plasmids for downstream applications

Figure 2 Polymerases chain reaction material and steps [5]

In order to check whether the PCR process worked, gel electrophoresis is used to visualize the fragment of the amplified DNA Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge[6] During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties The use of agarose gel electrophoresis revolutionized the separation of DNA Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-ast wells in the gel and a current applied The phosphate backbone [6]

of the DNA (and RNA) molecule is negatively charged, therefore, when placed in an electric field, DNA fragments will migrate to the positively charged anode Because DNA has a charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of their molecular weight The leading model for DNA movement through an agarose gel is "biased repetition", whereby the leading

edge moves forward and pulls the rest of the molecule along The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of the DNA molecule; 2) agarose concentration; 3) DNA conformation; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer After separation, the DNA molecules can be visualized under UV light after staining with an appropriate dye Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another All DNA molecules have the same amount of charge per mass Because of this, gel electrophoresis of DNA fragments separates them based on size only Using electrophoresis, we can see how many different DNA fragments are present in a sample and how large they are relative to one another The absolute size of a piece of DNA is also determined by examining it next to a standard ladder made up of DNA fragments of known sizes[6] DNA electrophoresis, particularly SDS-PAGE, is widely used for the separation and visualization of DNA fragments and proteins In this study, DNA electrophoresis was used to visualize the size of the amplified DNA fragment and to assess the success of the PCR amplification The purpose of this technique was to confirm the size and quantity of the amplified DNA fragment

Figure 3 An image of gel electrophoresis

III MATERIALS AND METHODS

3.1 Materials

In this experiment, substances such as LB agar, LB broth, ampicillin, and EDTA were required for manufacturing E coli competent cell JM109, which was kept in an ice bath The transformation task requires the plasmid, Tris pH 8.3, glycerol, and TSB broth A teaching assistant then treated the cell pellet of JM109 with the plasmid pBR322 and kept it in an ice bath to manufacture the plasmid During this experiment, buffers P1, P2, N3, PB, PE, and EB were also utilized The next step in this project was PCR and DNA electrophoresis, which necessitated the use of a PCR buffer and a TAE running buffer As a result, chemicals such as Tris-base, CH3COOH, 0.5M Sodium EDTA, and nanopure H2O were required for the manufacture of running buffer, while 10X Master Mix, forward and reverse primers, and autoclaved H2O were required for another Additionally, several reagents were prepared for grams taining such as crystal violet, distilled water, iodine, alcohol, safranin, and negative and

50 μL positive plates, LB medium without Ampicillin, LB with 100 μg/mL with Ampicillin for O.N Distilled water, micropipettes (P20, P200, and P1000), tips, Eppendorf, alcohol light, inoculation loop, L shape spreader, Petri plates, and ethanol spray bottle were used in all studies This method also included the use of an analytical balance, a water bath, a centrifuge machine, a PCR machine, a microplate reader, an autoclave, a microscope, slides, cover slips, and Kim Wipe papers, a QIAprep Spin Kit, and electrophoresis units

3.2 Methods

Before beginning this study, 2.5g LB broth (Miller Luria Bertani Broth) was weighed and mixed with 100 mL of distilled water to make LB medium for E coli component cell JM109

LB media was thoroughly dissolved in the stir plate at 75 C using a magnetic stir bar Then, o

2.5 g of LB and 15 g of agar were mixed together in 100 mL of distilled water In addition, a stir bar was inserted and autoclaved The solution was then chilled for 15-20 seconds before adding 2 mL of ampicillin Two bottles were labeled and kept at 4 degrees Celsius The job was broken down into four tasks:

3.2.1 Observation of bacteria

To observe the bacteria in this section, two approaches were used: heat-fixing and gram staining The glass slides were first cleaned and labeled In the center of the slide, a drop of

distilled water was placed Following that, just 10 μL of the bacteria from the positive plate were collected and placed on the slide using the inoculation loop in four plates An alcohol light was used to dry the sample on the glass slide For gram staining, 3-5 drops of crystal violet were applied to the sample and allowed to sit for 1 minute before washing with distilled water Several drops of Lugol iodine were applied to the stain and allowed to sit for 30 seconds A destaining solution was also used to wash the material A few drops of Safranin O were also added to color it, and the sample was left for 60 seconds Finally, the material was examined under a microscope at 40X using aluminum oil

3.2.2 Transformation

The experiment was conducted near an alcohol lamp, with all items used being sterilized Firstly, 20 μL of E coli competent cell JM109 was mixed with 1mL of 0.1M CaCl in the 2

eppendorf After that, it was placed in ice for 20 minutes and centrifuged at 4°C with 4000 rpm for 10 minutes to collect pellets 1μL of plasmid pBR322 and 20 μL of broth were added respectively into the eppendorf containing the pellet Subsequently, they were mixed gently and incubated in ice at 4°C for 30 minutes before being incubated at 42°C for 30 seconds After the sample was then placed on ice for 2 minutes, it was incubated at 37°C for 60 minutes Lastly, 1 mL of TSB broth was added and shaken

After completion of the heat-shock process, the sample was diluted with different factors (10μL, 50μL, and 100μL) to make three positive plates with the L-shape spreader In addition, 50μL of negative cells were dispersed to produce a negative plate for negative controls Finally,

it was incubated overnight at 37°C

3.2.3 Plasmid preparation

The experiment was conducted near an alcohol lamp, with all items used being sterilized A sterilized inoculation loop was used to collect cells from a JM109 colony on a 100 μL (+) ampicillin petri dish These cells were then transferred into a 1.5 mL Eppendorf tube containing

50 μL of LB agar (+) ampicillin Next, 250 μL of buffer P1 was added to each pellet using a

1000 μL pipette, resuspending the cells and transferring them to a microcentrifuge tube Subsequently, 250 μL of buffer P2 was added, and the tube was gently inverted 4-6 times until the solution became viscous and slightly clear, which took no more than 5 minutes Then, 350

μL of buffer N3 was added, mixed gently 4-6 times to stabilize the solution without causing precipitation, resulting in a cloudy appearance The mixture was centrifuged for 10 minutes at

13,000 rpm, and the supernatant was transferred into a QIAprep spin column using a pipette The QIAprep spin columns were cleaned with 300 μL of buffer PB and centrifuged at maximum speed for 1 minute, discarding the flow-through The process was repeated by centrifuging for 1 minute at 14,000 g, and the flow-through was discarded The QIAprep spin column was washed again with 500 μL of buffer PB and centrifuged for 1 minute at 14,000 g, discarding the through This step was repeated with 750 μL of buffer PE, and the flow-through was discarded The QIAprep column was then placed in a clean 1.5 mL microcentrifuge tube and centrifuged for an additional minute at 14,000 g to remove any remaining wash buffer Each QIAprep spin column was filled with 50 μL of buffer EB, left to sit for one minute, and then centrifuged to elute the DNA The supernatant was kept, and the pellet was discarded

3.2.4 PCR and DNA electrophoresis

To begin, the TAE buffer was made by combining Tris - base, CH3COOH, and 0.5M sodium EDTA and nanopure H2O, whereas PCR buffer was made by combining 25 mL 10X Master Mix, 10μL of 2μM of each primer (forward and reverse), and 5μL autoclaved dH2O Following that, the PCR buffer volume was divided into 1 PCR tube The template was then inserted into 1μL before running for 1 hour The thermocycler was loaded with a PCR tube, which was amplified for 30 cycles A TA carried out the preceding steps Simultaneously, an agarose gel was created by combining 0.75 grams of agarose and 50 mL of 1X TAE buffer The PCR tubes were added to the thermocycler and run 15 cycles of amplification at 95°C for 15 seconds, 59°C for 30 seconds, and 72°C for 1 minute.The 1X TEA running buffer was prepared by: 20

mL of 50X TEA buffer should be diluted with 1μL of nanopure water and stored in a 1μL glass bottle After that, the agarose gels were prepared: A 250 mL flask contained 0.75 grams of agarose and 50 mL of 1X TEA buffer After melting the agarose completely in the microwave, added 5μl of Gelred Nucleic Acid Stain, stirred well, and poured the mixture into the gel rig Let the gels sit for about 15 minutes, then the comb was removed and the gel was loaded into the electrophoresis chamber The electrophoresis chamber was set up close to an electrical outlet The DNA ladder was defrosted on some ice Aliquoted roughly 1μL of 6X sample buffer

to parafilm using a 10μL pipette and the appropriate tips 5μL of PCR samples should be mixed with a 10μL pipette Each well was filled with 5μL of each sample and the DNA ladder Finally, the gel was run for 30 minutes at 150V The gel was taken out of the chamber and soaked in

100 mL of 1X TAE and 0.5 g/mL EtBr for 30 minutes to Ethidium Bromide staining