Essay: Benefits of Molecular Biology Through Biotechnology Experiments



Molecular Biology

By Emani Williams

Biology 1100

November 20, 2018

Lab Partners:

Jada Hart

Rebecca Ferguson

Wilson Hawkins

Introduction

Biotechnology has been used by humans for millennia, including the use of fermentation, the maturation of cheese, etc. but it has more recently helped to develop many medical advancements that are capable of aiding in the sustenance of human life, amongst benefits to other organisms. Biotechnology is a type of technology that harnesses the potential of organisms’ biological systems, to develop products in the medical, agricultural, and research industries, etc. Biotechnology is helping to “heal, fuel, and feed the world.” The use of biotechnology helps to research behaviors of infectious diseases, resulting in the reduction of the spread of these diseases, decreases the amount of greenhouse gas emissions by increasing the use of biofuels, decreases water and waste use and production, increases the number of crops produced with less work, and much more (Biotechnology Innovation Organization, 20). There are multiple techniques associated with biotechnology, including but not limited to, Recombinant DNA, gene editing, and transformed organisms.

The purpose of this experiment was to use artificial transformation to successfully transform E. Coli cells that were exposed to plasmid and grow luminescent colonies on our experimental control plate (+ plasmid, LB, AMP), and calculate the transformation efficiency of colonies on our experimental plate. If we add plasmid, LB, and AMP all on one plate, then we would observe some growth of colonies on our experimental plate, and the plate should be able to glow as well. We expected to see some growth (not as much as we expected to see on our positive control plates), and the ability to glow on our experimental plate. The only plate that we did not expect to culture E. Coli colonies was the plasmid negative plate with LB and AMP.

MATERIALS AND METHODS

For this experiment, we first labeled two tubes “+ plasmid” and “- plasmid.” After labeling the tubes, we were to add 250 L of cold Calcium Chloride to each tube, to open up the cell membrane in the presence of heat to accept new DNA. It was important to keep the tubes upright and to suspend the cells using an inoculating tube. The tubes had to be placed on ice for 15 minutes after the TA added the plasmid to the positive plasmid tube. While we waited for the tubes to cool, we labelled our plates.

The positive control plate contained the plasmid and Luria Broth (LB), the negative control plate only contained the LB and Ampicillin (AMP), the experimental control contained the plasmid, LB, and AMP, and another plate only contained the LB. After allowing the tubes to sit on ice, we had to take the tubes straight off the ice and stick them into a 42C water bath for 90 seconds. While in the water bath, we had to agitate the tubes and after 90 seconds, we transferred the tubes back to the ice for another 60 seconds. We then used a sterile transfer pipet to add 250 L of Luria broth and mixed the LB with cell suspension by tapping the tubes with our fingers. We let the tubes come up to room temperature for 10 minutes.

After reaching room temperature, we placed 100 L of bacteria on each designated plate using a sterile transfer pipet (cell suspension should have reached the first graduation on pipet tip). The solution in the tube that contained the plasmid was placed in the plates that were plasmid positive and the solution in the tube that lacked the plasmid was placed in the plates that were plasmid negative. We used the hockey stick technique, which required us to rotate the plate while holding the hockey stick still, instead of moving the hockey stick around to spread the bacteria. We then left the plates to sit face up to allow the liquid to soak into the gel, then we flipped the plates face down, taped them, and placed them in an incubator set at 30C, and allowed them to incubate for 36 to 48 hours.

RESULTS

Table 1.1 Transformation Efficiency of Plates with and Without the Presence of Plasmid and AMP in a 48-Hour period.

Colony Number Transformation Efficiency Number

Positive Control

-Plasmid, LB 11 colonies N/A

Negative Control

-Plasmid, LB, AMP N/A N/A

Positive Control

+Plasmid, LB 25 colonies N/A

Experimental plate

+Plasmid, LB, AMP 3 colonies 1.5601 x 10²

Transformation efficiency only applies to competent cells (cells that are able to take up DNA).

Figure 1. Experimental Plate (+Plasmid, LB, AMP). This plate only grew 3 colonies.

Although it is capable, it did not develop produce a glow.

Figure 2. Positive Control Plate (+Plasmid, LB). This plate grew about 25 colonies and 1 lawn.

Did not develop the ability to glow.

Figure 3. Positive Control Plate (-Plasmid, LB). This plate grew about 11 colonies.

Did not develop the ability to glow.

Figure 4. Negative Control Plate (-Plasmid, LB, AMP). No colonies or lawns grew on this plate.

Also, did not develop the ability to glow.

As seen in figures 2 and 3, both positive control plates ( Plasmid, LB) were able to grow copious amounts of colonies, which was the expected result for the positive controls. The positive control that had the plasmid was able to grow the most colonies in the 48-hour incubation period (Table 1.1). The positive control without the plasmid grew the second greatest number of colonies (Table 1.1). The experimental plate did not grow many colonies, and only produced 3 colonies (Figure 1, Table 1.1).

Although the ability to glow was expected for the +Plasmid and LB plate and the +Plasmid, LB, and AMP plate, neither of the plates proved to be bioluminescent. We determined the total mass of our plasmid to be 0.1g (20L/0.005g/L). The total volume of our cell suspension was 520L, fraction speed was 0.1923L (100/520). The mass of the plasmid spread in our cell was 0.01923g (0.1g/0.1923). Finally, our Transformation efficiency was calculated to be 1.5601 x 10² (3/0.01923g).

DISCUSSION

The purpose of this experiment was to use artificial transformation to successfully transform E. Coli cells by growing luminescent colonies on our experimental plate (+ Plasmid, LB, AMP), and determine the transformation efficiency of cells on the experimental plate. My hypothesis was supported in terms of growth, but because of the lack of luminescence, my hypothesis must be rejected. In this experiment, we were selecting for a plasmid that contains the gene for ampicillin resistance. Without the bacteria developing the AMP resistance gene, the bacteria on the plate will prove to be non-viable. The phenotype of the bacteria indicates that the bacteria have successfully been transformed with the Plasmid because although the bacteria did not develop the ability to glow, they grew on the Ampicillin proving to be viable and received the AMP resistant gene (Dr. Rapoza). The first plate that would be able to prove the transformation occurred would be the experimental plate (+Plasmid, LB, and AMP), which can be supported with our calculated transformation efficiency of 1.5601 x 10². Transformation is so important because it allows transformed cells to adapt to new environments. Because of this adaptation, we are able to perform DNA cloning, replicate specific human proteins to allow for research on diseases, and allows DNA to genetically modify bacterium and other cells (Science Learning Hub, 2007).

WORKS CITED

Alston, Bailey. “Molecular Biology.” Biology 1101 Lab. Clemson University, Clemson, SC. November 2018. Lecture.

“Bacterial Transformation.” Science Learning Hub, Nov. 2007, www.sciencelearn.org.nz/resources/2032-bacterial-transformation.

Dr. Rapoza, Maria, et al.” Fundamentals of Molecular Biology.” Handout. Carolina Biological Supply Company. N.d. Print.

“Healing, Fueling, Feeding: How Biotechnology Is Enriching Your Life.” BIO, www.bio.org/healing-fueling-feeding-how-biotechnology-enriching-your-life.

“Transformation of the Bacterium E. Coli Using a Gene for Green Fluorescent Protein.” APS, 0AD, www.apsnet.org/edcenter/K 12/TeachersGuide/PlantBiotechnology/Documents/TransformationEcoliBacterium.pdf.

Urry, Lisa A, et al. “Lab Topic 5: Cellular Respiration and Fermentation.” Principles of Biology Laboratory, 9th ed., Pearson Education, 2017, pp. 118–123.

“What Is Biotechnology?” BIO, www.bio.org/what-biotechnology.