Essay: Transform Escherichia Coli Through Bacterial Transformation with Plasmids pUC18 and lux



Abstract

Gene expression can be described as the process in which information from a gene leads to the synthesis of a functional gene product. The genes in DNA can be manipulated and engineered through the movement of DNA from one organism to another to carry a particular characteristic which the other organism might need. In this experiment Escherichia coli is tested to see the results of pUC18 and lux, 2 antibiotic resistant plasmids, on bacteria in an ampicillin rich medium. The growth observed can either be lawn, colonial, or none, which show the range in which the bacteria physically grew. Calcium Chloride was used in this experiment to increases the ability of the E. coli cell to incorporate plasmid DNA allowing it to be genetically transformed. In addition to the calcium chloride, the heat shock technique was used in this lab to alter membrane fluidity and creating pores in the plasma membrane which allowed the plasmid DNA to enter the bacterial cell. Through this experiment the ability to genetically manipulate the E. coli in order for it to become resistant to ampicillin, which would otherwise kills bacteria, was seen. The data obtained matched other researcher’s data and reliability of this experiment. Bacterial transformation has influenced how medicines can be produced in order to find more cures for many types of diseases. DNA carries the ability to be successfully transformed in all different types of organisms, this is because of the versatility of genetic codes within many organisms.

Introduction

DNA is the foundation of all living things; it is the blueprint of life and without it life would not be possible. In order for transcription to occur and for DNA to be interpreted correctly, the DNA is copied to a new molecule of messenger RNA. This mRNA carries the information needed for protein synthesis. It serves as guide for protein synthesis during translation; the mRNA then translates the DNA into proteins. These proteins can be described as the language of the cell and are responsible for determining the physical and biochemical properties as well as the function of the cell (Alberte et al,. 2012).  Once transcription has occurred and the RNA has left the nucleus it can be translated into a polypeptide sequence, a process termed translation. These processes happen differently in prokaryotes than they do in eukaryotes since prokaryotes don’t contain a nucleus. Since prokaryotes lack a nucleus it makes the process of bacterial transformation less complex, as opposed to in eukaryotes. The most known form of DNA is nuclear DNA however, there are other forms in which DNA is also found, including plasmids. Plasmids are small, circular DNA molecules that exist in the nuclei region of many bacterial species. Although not  necessary for survival of the host, plasmids can give advantages needed for the bacteria to survive and reproduce. Plasmids provide genetic advantages such as antibiotic resistance. Gene expression is the process in which the possession of information from a gene leads to the synthesis of a functional gene product. The genes in DNA can be manipulated and engineered through the movement of DNA from one organism to another  to convey a particular characteristic (Mason et al,. 2014). Quorum sensing is the regulation of gene expression when fluctuation in cell population density is seen. In bacterial, quorum  sensing produces and releases chemical signal molecules called autoinducer that increase in concentration as cell density increases or decreases (Chen et al., 2002).

 The purpose of bacterial transformation is to introduce a plasmid to the bacteria. After the plasmid has been introduced the bacteria will amplify the plasmid and make large quantities of it. The insertion of genes will make E. coli resistant to the antibiotic ampicillin and in doing so will allow it to continue to grow.

The insertion genes that will make Escherichia coli, a bacterium usually found in the human gastrointestinal tract, resistant to ampicillin and to possibly glow. This process of bacterial transformation was discovered by Frederick Griffith, a physician from London, in 1928 with the transformation of Streptococcus Pneumoniae (Lorenz 1994). E. Coli is known to have strains, a genetic variant of a microorganism, that are pathogenic for infections and diseases, such as urinary tract infection, diarrhea and pneumonia.  (Levine et al., 2003). E. coli was formerly thought to be incapable of developing competence however, with the use of calcium chloride the cells develop the ability to become competent and accept plasmids (Baur et al., 1996). The temperature which were tested in this experiment, that the bacteria was incubated in did not affect the growth but rather the rate of transformation. Heat-shock transformation is when plasmids are incorporated into chemically-competent cells and made permeable by the use of heat shock and calcium chloride solution. The sudden increase in temperature creates pores in the plasma membrane which allows the plasmid DNA to enter the bacterial cell and make the plasmid have a higher chance of being incorporated (Inoue et al., 2003).

E. coli is a commonly used bacteria in biotechnology because of its rapid growing ability and its short reproduction time. (Alberte et al,. 2012).

In this experiment the competency of the bacteria cells will increase with the introduction of the plasmids, pUC18 and lux, to the E. coli petri dish. The plasmid will make the cells develop antibiotic resistance and in doing so will promote cell growth. If the plumed fails to work then it can be determined that the bacteria’s cell did not take in the plasmid. The bacteria given the LBc  and LBlux will show lawn growth while the LB/Ampc  and LB/Amplux will have colonial growth. The LBNP and LB/AmpNP will serve as the positive and negative controls.

Method

Before starting the lab some step were completed by the instructor. These steps included placing a vial of CaCl2 and a tube of the E. Coli into an ice bath, transferring 590μL CaCl2 solution to the 50μL of the bacteria, mixing the tube with the index finger to mix the solution, and incubating the cells in ice for at least 1o minutes. After the 10 minute incubation period the cells can be referred to as competent because they can then take up DNA. The control plasmid, pUC18, and the plasmid lux will introduced to the E. coli; this will allow transformation to occur and the bacteria will take in the gene chosen. The control plasmid will give antibiotic resistance and the plasmid lux will give off both antibiotic resistance and bioluminescence (Alberte et al,. 2012). Label one Eppendorf tubes as “C” and one as “lux”, for the plasmids, and place them in an ice bath. Then, using a sterile micropipette 5 µL of the control plasmid should be added into the tube labeled “C” and 5 µL of the lux plasmid into the tube labeled “lux”. All tubes should be kept in the ice bath until instructed to take out.

Results

Figure 1. Bacterial Transformation of Escherichia coli using Lux and pUC18 plasmids either showing or not showing Ampicillin Resistance.

In Figure 1, the petri dishes all show the bacterial transformation of Escherichia coli when exposed to lux and pUC18 plasmids showing either no ampicillin resistance or ampicillin resistance. This ultimately shows whether the bacteria took in the plasmids or not. In the first and fourth pictures, LBc and LBlux, lawn growth can be seen with the exception of a few colonies. The  LB/Ampc and LB/Amplux  show colonial growth because although the plasmid was able to allow some of the e. coli to take in the plasmid in order to grow  and reproduce, it was exposed to the ampicillin causing a lot of the bacteria die. Finally there’s the  LBNP and LB/AmpNP , the control groups, which showed what a positive or negative result would look like; these two petri dishes minimized false negatives and false positives within the experiment.

Transformation Efficiency for LB/Amp𝐶
1. Amount DNA (µg) = Concentration of DNA (µg/µL) x Volume of DNA (µL)

0.005 𝜇𝑔/(𝜇𝐿) x3 𝜇𝐿 = 0.015 𝜇g of DNA

2. 𝑇otal volume (𝜇𝐿) = amount (𝜇𝐿) of plasmid + amount (𝜇𝐿) 𝑜𝑓 𝐿𝐵 + amount (𝜇𝐿) of cell suspension

3𝜇L 𝑜𝑓 𝑝𝑙𝑎𝑠𝑚𝑖𝑑 + 275𝜇L 𝑜𝑓 LB + 70 𝜇𝐿 𝑜𝑓 𝑐𝑒𝑙𝑙 𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛  = 348 total volume (𝝁𝑳)

3. Fraction of DNA spread =  Volume (𝜇𝐿) spread on 𝐿𝐵/𝐴𝑚𝑝c plate / Total sample volume (𝜇𝐿) in control DNA tube

275(𝜇𝐿)/348(𝜇𝐿) = 0.790 fraction of DNA spread

4. Total amount (𝜇𝑔) of DNA = (𝜇𝑔) of DNA x fraction of DNA spread

(0.015)(0.790) = 0.1185  μg of DNA

5.  Transformation efficiency = Total number of colonies on the LB/Ampc plate/ Total amount of DNA spread of the LB/Ampc plate

287/0.1185 = 2,421.94 transformants/ μg

Calculation Equations 1: Transformation Efficiency for LB/AmpC

Transformation Efficiency for LB/Amplux 
1. Amount DNA (µg) = Concentration of DNA (µg/µL) x Volume of DNA (µL)

0.005 𝜇𝑔/(𝜇𝐿) x3 𝜇𝐿 = 0.015 𝜇g of DNA

2. 𝑇otal volume (𝜇𝐿) = amount (𝜇𝐿) of plasmid + amount (𝜇𝐿) 𝑜𝑓 𝐿𝐵 + amount (𝜇𝐿) of cell suspension

3𝜇L 𝑜𝑓 𝑝𝑙𝑎𝑠𝑚𝑖𝑑 + 275𝜇L 𝑜𝑓 LB + 70 𝜇𝐿 𝑜𝑓 𝑐𝑒𝑙𝑙 𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛  = 348 total volume (𝝁𝑳)

3. Fraction of DNA spread =  Volume (𝜇𝐿) spread on 𝐿𝐵/𝐴𝑚𝑝𝑙𝑢𝑥 plate / Total sample volume (𝜇𝐿) in control DNA tube

275(𝜇𝐿)/348(𝜇𝐿) = 0.790 fraction of DNA spread

4. Total amount (𝜇𝑔) of DNA = (𝜇𝑔) of DNA x fraction of DNA spread

(0.015)(0.790) = 0.1185  μg of DNA

5. Transformation efficiency = Total number of colonies on the LB/Amplux plate/ Total amount of DNA spread of the LB/Amplux plate

94/0.1185 = 793.25 transformants/ μg

Calculation Equations 2. Transformation Efficiency for LB/Amplux

The calculation prove consistent with what was expected of the treatments however they are considered low when compared to what is viewed as effective cell competency. The results although not matching the high efficiency level of what scientist deemed good transformation efficiency of E. coli it still showed positive results. Even though 2×108 to 1011 , are deemed viable numbers for the efficiency of the cell any positive number shows that the bacteria took in the plasmid and was able to reproduce and pass on the antibiotic resistant genes. Transformation efficiency is used to measure whether the plasmid was successfully incorporated into the bacterial cells or not.

Table 1. Results, Escherichia coli when either exposed to either no plasmid, lux or pUC18 plasmids with or without ampicillin.

Table 1 shows the results that were observed for all the treatments and their yields. This data also shows that the experiment yielded the expected results and thus proved the viability of the experiment. Overall, the experiment was successful and showed that the E. Coli when given a food source will grow normally and show lawn growth, the same could be said about the bacteria given a plasmid while not being exposed to ampicillin; this is because the bacteria does not need the plasmid and will not have to use it. However, when the E. Coli is exposed to the ampicillin it will need a plasmid in order to accept the antibiotic resistant genes which will then allow it to grow and reproduce; these both will show colonial growth because not all the E. coli will be able to take in the plaid and survive. Finally, E. coli in the presence of ampicillin and no plaid will die because it does not have the means to develop antibiotic resistance.

Discussion

In this experiment, we tested the growth of E. Coli when given a plasmid and in exposure to ampicillin. From the data collected it can be deduced that the experiment was successful because the results were conclusive with that of scientist who have studied the reaction of ampicillin resistant plasmids. The plasmid was taken in by the E. Coli and allowed it to develop antibiotic resistance.  Luria Broth serves not only as the food source for the E. coli in order to promote healthy growth in the bacterial cells but also displayed what the experiment would look like without the inclusion of the treatments. The bacteria given the LBc and LBlux had lawn growth while the LB/Ampc  and LB/Amplux had colonial growth; this is because the LBc and LBlux  were not introduced to the ampicillin and although they did not need the plasmids, they were unaffected by it. However, the LB/Ampc  and LB/Amplux showed colonial growth because even though they were affected by the ampicillin some took up the plasmids and were able to grow and reproduce. The LBNP served as the positive control and thus showed the expected lawn growth. On the other hand, the LB/AmpNP which served as the negative control and had no growth. The controls were used a reference point to look back to and see whether any changes occurred in the other treatments. A possible source of error in this experiment could have been contamination. Even though many precautions were taken to avoid these type of errors such as, using disposable pipettes and sterile tools, there is a chance that these tools were already contaminated beforehand. These types of errors are often times unavoidable. Another source of error could have come from the cell spreader since it had to be dipped in ethanol and passed through the flame of the ethanol lamp. If the spreader was too hot it could kill some of the bacteria, which is possibly why the LBc , LBlux, and LBNP had a few colonies rather than an even spread of growth.

In a study by B Baur, K Hanselmann, W Schlimme, and B Jenni Escherichia coli given the lux and pUC18 plasmids was able to exhibit colonies of bacterial growth when in the presence of either mineral or freshwater (Baur et al., 2001). In other studies on transformation of E. coli with plasmids these same results can be seen. Drug resistance was expressed in small fractions of the bacterial population that twas given the plasmid almost immediately after uptake of the DNA. However, this study reported that full genetic expression of resistance demands subsequent incubation in a drug-free medium before antibiotic challenge. Transformed bacteria gain a closed circular and transferable DNA species with the resistance and fertility traits of the parent bacteria’s R factor. Again, this shows that after the bacteria uptakes the plasmid it should be able to pass it in on to other generation and in so giving them a higher rate of survival. The plasmid containing an antibiotic resistant gene should allow the bacteria to survive even in the presence of antibiotics. This lab has many future prospects when it comes to real world application. Genetic modification is not limited to antibiotic resistance and bioluminescence. Organisms can be given plasmids in order to create all sorts of unique traits. Plants, for example, can be given plasmids so that they gain certain traits, such as resistance to extreme weather which will allow it to survive when other plants can not. This can lead to better crop yield and more food source for humans. Another way that the plasmid could be used is that the lux which codes for bioluminescence can be added to genes in fish, or another multi cellular organism to create glowing fish (Baur,. 1996). There are also many medical benefits to genetically engineering bacteria. Gene expression is used to profile cardiovascular disease and towards more research on cardiovascular diseases (Henriksen,. 2002). Scientist were able to genetically engineer E. coli in order to secrete proteins that have can block HIV from infecting the cells of other organisms. (Bacteria modified,. 2005) Genetic modification of bacteria has been done for many different reason and have had a groundbreaking impact on the world. In conclusion, the null hypothesis is failed to be rejected stating that there is correlation between a transformed bacteria and its ability to grow on an ampicillin rich medium. Since we fail to reject the null hypothesis we can reject the alternative hypothesis. Overall these results matched the hypothesis offered at the beginning and provide conclusive data that prove if bacteria is able to take in a plasmid it will be able to grow and reproduce.