Name: Mason Schafer
Name: Madison Bang
Name: Danae Bosch
Name: Katie Urquhart
Mr. Chow
Biology AP
Period 1
22 February 2018
Madison Bang, Katie Urquhart, Danae Bosch, Mason Schafer By typing yes here, we declare that we are the sole authors of all the writing presented below. We agree to submit our complete & accurate work to turnitin.com. We understand that cheating, plagiarism, sharing work or submitting the wrong work to turnitin.com will result in a 0% on this assignment with no redo opportunity. We take full responsibility for our decisions in AP Biology.
I. Title: The Investigation of Bacterial Transformation and Its Effects on Growth and Physical
Properties by the Use of the pGLO Plasmid and Biotechnology
II. Introduction & Objective:
Throughout this unit, we have studied all sorts of genetic mutations and changes. This unit, in particular, is the study of biotechnology. More specifically, how gene cloning and transferring of works. In this lab, we achieved this through plasmids. Plasmids are small circular molecules of DNA that contains genes that are not often transcribed in a bacterium. One can insert a gene of interest into a plasmid that will give the plasmid a certain trait. In this specific lab, we used bacteria known as E Coli. to determine how individual plasmids can be transferred into a bacterium. This process of adding recombinant DNA, DNA that is artificially formed by combining DNA segments from two or more different organisms, is also known as genetic modification. This is more commonly known in the agricultural world as GMOs. The central concept of the lab is to successfully transfer a few genes of interest into an E. Coli cell so that they can express arabinose metabolization, ampicillin resistance, and the GFP gene of glowing under ultraviolet light. This lab is to understand how genetic change and transformation can occur in bacterium in a real-life scenario.
In this lab, it is using the bacteria named E. Coli and genetically modifying its genes so that it can express the 3 traits. E. coli is in the stomach and intestines of all humans where it breaks down foods and helps the digest it. However, some forms of this bacteria can be harmful to the body and can cause diarrhea, breathing problem, and urinary tract infections. E. coli is commonly used in experiments all across the world as it was used in many of the early experiments in the past. In addition, E. coli has a relatively simple genome with only about 4,377 genes in its genome according to Google whereas humans have about 20,000 of just protein genes.
Besides the E. coli bacteria, the lab uses a transformation fluid (CaCl2), an ice bath, and an hot water bath to carry out the experiment. Transformation fluid is a mixture specifically made for labs that is sterile. The purpose of the transformation fluid is to add a fluid solution that will aid in the mixing of all the components in the micro-test tube. The ice bath is very basic and is only a cup of water with ice in it. The purposes of this is to freeze the lipids in the E. coli’s membrane in order to distribute the negative charge of the phosphate head. Lastly, the water bath is at 42 degrees celsius to act as a heat shock for the bacteria.
In order to insert the plasmid one first preps the vials for the bacteria and other fluids, transformation fluid and the LB broth. One of the vials has pGLO in it (+pGLO), and the other doesn’t (-pGLO), both are very important to the lab because the pGLO plasmid is the independent variable. After the vials are all set up, place them in an ice bath to stabilize the cell membrane before it has to be changed. Next, we give the vials a heated show to create a thermal imbalance otherwise known as a gradient. This allows easy access to the cell membranes inside the vials now have a draft from the gradient pulling in the plasmids. After a selected amount of time it takes for the heat shock, you immediately place the vials into the ice bath again after shaking the test tubes, to allow the cell membranes to close keeping the new plasmids of pGLO inside to the E. Coli cells. This is the beginning process of transformation. Along with the vials, we separate an even amount from the vials twice onto Petri dishes. These are the tests that we run: +pGLO; LB/amp, +pGLO; LB/amp/ara, -pGLO; LB/amp, and -pGLO; LB. LB is a nutrient broth that allows the colonies to grow, amp or ampicillin is an antibiotic that is supposed to kill E. Coli, and ara or arabinose is what allows the GFP operon to be enabled, this makes the arabinose the inducer. These four test then go into a climate controlled container to then be observed in the morning. The objective of this lab is to achieve transformation (Glowing colonies) through the act of genetic modification.
III. Variables & Control:
During the experiment, the independent, the variable that was directly changed to modify the results of the experiment, was the pGLO plasmid which was incorporated into the E. Coli bacteria. This is the independent variable because the plasmids containing this gene were directly added or held back from the E. Coli bacteria.
The dependent variable, the variable that reacted to the addition of the pGLO independent variable, was the number of colonies of E. Coli bacteria, which had its genetic makeup altered to exhibit specific traits. This was measured by counting the number of colonies that grew on the plates after being left to incubate overnight.
In the experiment, there were two plates that were used as control groups. These plates both contained LB broth while one had ampicillin. The bacteria placed into the controlled plates did not have the pGLO gene. These controls were used to in order to compare the activity on the plates with the same nutrient but with transformed bacteria or non-transformed bacteria. This control was highly important in order to see why we need ampicillin resistance, one of the genes inserted into the plasmid, in the bacteria since they die upon contact with ampicillin.
IV. Constants:
In the lab, there are some materials that stay the same, the constants. One constant was incubation period. This is where we kept the Petri dishes with the E Coli. bacteria in a temperature controlled climate overnight to recover and then be observed in the morning. A second constant is the use of LB broth. It supplied nutrients for the bacteria colonies to grow and was used in all four bacteria plates. The third constant is the procedure. During the lab, we followed the same procedures for each vial to make sure that each action was as close to the same as possible. If an action was done to one micro-test tube, it was immediately done to the other tube. The fourth constant is the time in the warm water and ice. Since the micro-test tubes were constantly next to each other in the foam rack, it was ensured that they spent exactly the same time in the ice bath or water bath.
V. Materials:
Two micro test tubes
Waterproof marker
Foam Rack
Sterile micropipette
Ice
Transformation Solution
Two Sterile Loops
Four Plates
LB Agar Broth
Ampicillin
Arabinose
PGlo Plasmid
UV light
E. Coli Cells
Water Bath at 42 C
LB Nutrient Broth
VI. Hypothesis:
If pGLO is combined with two colonies of E. Coli bacteria and two separate colonies of bacteria without the plasmid are allowed to incubate in LB agar, then the plates with +pGLO bacteria will become continue to grow in ampicillin and glow when introduced to arabinose while the untransformed plates will die when ampicillin is introduced, because the pGLO plasmid contains the genes required for ampicillin resistance and GFP, which can be regulated in cells depending on the presence of arabinose, and E. Coli contains three enzymes that can break down arabinose, enabling the GFP to be active.
VII. Procedures:
Label one closed micro test tube +pGLO and another –pGLO. Label both tubes with your groups number (e.g. G2). Place them in the foam tube rack.
Open the tubes and using a 100-1000 L micropipette with a sterile tip, transfer 250 L of the ice cold transformation solution (CaCl2).
Place both tubes on (into) the ice.
Use a sterile loop to pick up a single colony of bacteria from your starter plate. Be careful not to scrape off any agar from the plate. Pick up the +pGLO tube and immerse the loop into the CaCl2 solution (transforming solution) at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the solution. Use the 100-1000 L micropipette with a sterile tip and repeatedly pulse the cells in solution to thoroughly resuspend the cells. Place the tub back in the ice. Using a new sterile loop, repeat for the –pGLO tube.
CAUTION: Keep nose and mouth away from tip end when pipetting suspension culture to avoid inhaling any aerosol that might be created.
Examine the pGLO plasmid DNA solution with the UV lamp. Using a 1-10 L micropipette with a sterile tip, transfer 10 L of the 0.08 g/ L pGLO plasmid solution directly into the E. coli cell suspension in the +pGLO tube. Tap tube with a finger to mix. Avoid making bubbles in the suspension or splashing the suspension up the sides of the tube. Do not add the pGLO plasmid solution into the –pGLO tube.
Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the ice.
While the tubes are sitting on ice, label each of your four agar plates on the bottom (not the lid) with your group number and the date; then as follows:
Label one LB/amp plate +pGLO
Label the LB/amp/ara plate +pGLO
Label the other LB/amp plate –pGLO
Label the LB plate –pGLO
Following the 10 minute incubation at 0°C, heat shock the cells in the +pGLO and –pGLO tubes. It is critical that the cells receive a sharp and distinct shock. Using the foam rack as a holder, transfer both the +pGLO and –pGLO tubes into the water bath, set at 42°C, for exactly 50 seconds. Make sure to push the tubes all the way down in the foam rack so the bottom of the tubes with the suspension makes contact with the warm water. When the 50 seconds are done, place both tubes back on ice. For best transformation results, the change from 0°C to 42°C then back to 0°C must be rapid. Incubate the tubes on ice for an additional 2 minutes.
Remove the rack containing the tubes from the ice and place on the lab counter. With a 100-1000 L micropipette with a sterile tip, transfer 250 L of LB nutrient broth to the +pGLO tube. Close the tube and gently tap with your finger to mix. Repeat with a new micropipette tip for the –pGLO tube. Incubate each tube for 10 minutes at room temperature.
Use the 10-100 L micropipette with a sterile tip to transfer 100 L of the transformation and control suspensions onto the appropriate plates. Use a sterile tip for each of the four transfers.
Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the agar by quickly skating the flat surface of the sterile loop back and forth across the plate surface. Do not poke or make gashes in the agar. Allow plates to set for 10 minutes.
Stack your plates and tape them together. Place the stack upside down in the 37°C incubator for the next day. (We might have to use a homemade incubator…)
VIII. Results and Observations:
Throughout our experiment in bacterial transformation, we observed both qualitative and quantitative information. These observations allowed us to further see out the outcome our lab. From the procedural steps to the ending results, both quantitative and qualitative observations were made. By taking note of different things we saw, our group is able to fully analyze what happens during our experiment and how different observations match with the different occurrences.
The qualitative observations are notes taken during the course of an experiment pertaining to the physical attributes seen from the procedure, specimens, and results. While going through the procedure steps, we noticed how the micropipettes obtained air bubbles while we were taking in measurements of transformation solution. In addition, our team noticed how time effective we had to be. From incubation to heat shock to recovery, understanding the time crunch was necessary to the efficiency of our lab. The best method, which our group was able to utilize, was to prepare for a procedural step beforehand and to complete the laboratory questions while incubation occured. This gave us the most time to effectively carry on our experiment. In regards to the specimens throughout the experiment, we noticed an odor when handling them throughout the second day, similar to the smell of old chicken soup. The class was cautioned to take extreme care when handling the bacterial colonies, as E. coli is harmful to the human body. Our group found that the most effective way of using a sterile technique was utilizing rubber gloves and thoroughly washing our hands. In transition to the observations of our results, we saw how our bacterial colonies were smaller in size compared to other groups. Nevertheless, these colonies were easy to count because they were more spread out. The results with 18 colonies of bacteria in the transformed bacteria located in a plate of LB, ampicillin, and arabinose were simply by chance because this number was dependent on which cells took in the pGLO plasmid. Seen in this plate, the bacterial cells would both grow and glow due to the plasmid resistance to ampicillin and protein regulator of GFP. With the other plate of transformed bacteria, we saw more colonies but no glowing because of the environment changes. This dish was similar to the prior dish mentioned above, except for the lack of arabinose in the environment solution, thus disabling the ability to glow. With our plates of -pGLO, we saw results of polar opposites. When ampicillin was inserted into the petri dish, there was no growth due to the lack of the pGLO plasmid to resist the antibiotic. Without ampicillin, there was normal growth and full coverage of the colonies because there was simply nutrients and no limitations/regulator.
The quantitative data are calculated from the numerical data that is obtained throughout the course of the experiment. After concluding the experiment the transformation efficiency can be calculated with all of the information gathered during the process. The transformation efficiency states how many colonies will become resistant to the antibiotic per microgram of pGLO combined with the bacteria. In the experiment 10 microliters of pGLO was used and it had a concentration of 0.08 ug/uL, meaning that the total mass of the pGLO added to the bacteria was .8 uL. After combining 250 uL of nutrient broth, 250 uL of transformation solution, and 10 uL of pGLO into a micro test tube the total volume of the cell suspension came out to be 510 uL. Of this entire suspension only 100 uL of it was actually spread onto the agar plate, meaning that the fraction of suspension used was .196 uL. Using this information the total mass of the pGLO spread can be found by multiplying the mass of the pGLO along with the fraction of suspension, giving it a mass of .156 uL. After the bacteria with pGLO added to it was left to incubate overnight, 18 green fluorescent colonies were in the LB agar. To finally determine the transformation efficiency the number of colonies in the agar is then divided by the mass of pGLO spread, determining that it has a transformation efficiency of 114.75 uL.
Placing tubes in a First Incubation Adding in E. Coli Mixing the E. Coli into foam rack the micro-tubes
Heat Shocking the Bacteria The Glowing of the Transformed
Bacteria Colonies
IX. Data:
Overall, this experiment of bacterial transformation utilized four plates: two containing transformed bacteria and two containing non-transformed bacteria. We have seen a large difference in how the bacteria have grown in the plates depending on their coding and environment.
In the first plate, we had transformed bacteria with the pGLO plasmid and LB/ampicillin nutrients in the bottom of the plate. After incubating for 24 hours at 37 C, we observed that the bacterium had grown even though there was ampicillin in its environment. We had counted approximately 58 colonies in total that had grown on top of the agar. The bacterium grew in off-white spots, not covering the entire surface, and the sizes of the spots range from the tip of a needle to the thicker back of a needle. The reason that the bacterium did not cover the whole surface is that only a few of the thousands of bacteria actually accepted the plasmid and those that did not transform, died due to the ampicillin in the agar mixture. The single bacterium that accepted the plasmid then started reproducing during the incubation period to start new colonies.
In the second plate, we had transformed bacteria with the pGlo plasmid and LB/ampicillin/arabinose nutrients in the bottom of the plate. When taken out after the 24 hours of incubation, we counted 18 colonies of transformed bacteria. When the UV light was shown on the colonies, they glowed bright green. The reason that this plate glowed yet the first plate did not is that the second plate contains the arabinose sugar which acts as the co-repressor to remove the repressor protein on the GFP operator so that the glowing gene can be expressed.
On the third plate, when had non-transformed E. Coli bacteria and ampicillin/LB. Naturally, ampicillin kills bacteria by inhibiting the synthesis of their cell wall which leads to automatic cell death. Therefore, because the E. Coli did not have the plasmid with ampicillin resistance and died during the incubation phase. We saw no growth on the plate since they all died.
On the fourth and final plate, we had non-transformed E. Coli bacteria and LB broth. Since there is nothing inhibiting the growth of the E. Coli but only nutrients to support the growth, the bacteria grew all over the surface and the entire plate was covered. The bacterium did not form little groups in the shape of dots as all the other plates had but instead was a single layer even distributed.
Our data turned out exactly how we had hypothesized it would and was extremely interesting to see. Being able to understand what happened and why certain events took place due our background knowledge of transformation made the lab more interactive and intriguing. For example, we knew that not all bacterial cells could take in the pGLO plasmid, depending on its stage in growth. This is why, with our dishes of transformed bacteria, there was not full coverage of the plate (smaller number of colonies). Depending on the environment, the bacteria would react in a certain way due to the traits encoded in the plasmid (when accepted). On the other hand, the untransformed bacteria was able to grow when it was simply in broth full of nutrients. Nevertheless, without plasmid being available and accessible, there was no gene to combat ampicillin in the petri dish, which is why there was no growth in that plate of untransformed bacteria.
Plate Type
Number of Colonies
LB w/ -pGLO
Full coverage
LB/AMP w/ -pGLO
No growth
LB/AMP/ARA w/ +pGLO
18 colonies
LB/AMP w/ +pGLO
58 colonies
X. Discussion:
How did your group calculate transformation efficiency? Explain the steps you took, your answer and explain what that means.
The calculation of transformation efficiency is key to the basis of this entire experiment of transformation bacterial cells. One uses transformation efficiency to calculate the growth rate of transforming antibiotic resistant colonies per microgram of pGLO used. In our experiment, this merely means that for every microgram there is approximately 1.1475 ✕ 10^2 μL of transformed bacteria. In the lab, we concluded that there were approximately 18 green fluorescent colonies. After determining the number of colonies, we proceed to find the total mass of pGLO used which was 0.8 μL (concentration) ✕ 10 μL (volume) resulting in 0.8 μL. Before we continued with the calculations, we had to find the total volume of the cell suspension prepared; this was just simply adding 250μL, 10μL, and another 250μL giving us a total volume of 510. We used the 510 μL to calculate the fraction of suspension which is the number of μL ✕ total volume prepared. In other words, 10/51. Using our first calculation of 0.8 μL and 10/51 we were able to determine the total mass of pGLO spread, which was about .157 μL. Finally, the last calculation was the number of colonies observed (approx. 18) ÷ total mass of the pGLO spread (approx .157) making the transformation efficiency 1.1475 ✕ 10^2 μL.
When calculating transformation efficiency, it is important to understand that transformation is limited only to those cells in the stationary phase of growth. A sample of “competent” cells is usually saturated with small amounts of plasmid, such as our pGLO; but any excess DNA may actually interfere with the process of transformation. Simply increasing the amount of plasmid exposed to the bacterial cells will not necessarily increase the amount of transformed DNA. The action of bringing in the plasmid is solely dependent on whether or not the cell can take it in. Thus, the transformation of our team’s bacterial is based on how effective and correct our following of procedures were, as correct intubation, heat shock, and recovery will allow for the plasmid to be taken in.
How could we “extend/further this lab”? Where can we go from here?
With a lab surrounding the subject of bacterial transformation, there are endless possibilities. To further and continue the lab, we could do gel electrophoresis to validate the bacterial DNA had been transformed. To make this happen, you would use gel electrophoresis on the +pGLO and -pGLO colonies. Then to validate that there has been a transformation, you would check the four bacterial sample’s DNA ladders. Looking at these ladders, the two that are negative should be the same, and the two that are positive should be the same. However, the positive ladders and the negative ladders should not be the same. If the experiment ended up the same as expected, then you know that there has been a change to the bacterial DNA’s amount of restriction enzymes, because of the addition of the pGLO plasmid to the bacterial DNA, would produce more and different restriction sites.
The experiment could even move away from bacterial cells. Similar to the learning of using biotechnology for medical purposes, what if we were able to inject specific DNA sequences to aid one in the recovery or healing of a disease? Using biotechnology, an experiment could be conducted to first analyze the DNA sequence that is faulty and what aspect of it is being expressed phenotypically. From there, plasmids, or foreign DNA in general, could be created to tackle the faulty genes. Moreover, how could we use this subject of transformation to genetically modify certain plants and produce? For our benefit, we could combat problems with growing and nurturing for produce by developing insertions and other methods around the basis of transformation and conjugation. As one can see, both smaller and larger aspects of this experiment can be altered to further continue the project, even for the better of the world.
XI. Analysis of Data and Conclusion:
The purpose of this experiment was to see if we could introduce a plasmid with foreign DNA into an E. coli cell, giving the bacterium ampicillin resistance, the ability to metabolize arabinose, and the ability to glow under UV light. Another purpose was to future our learning as we had to recall how genes are expressed in prokaryotes since the GFP, the glowing gene, had an inducible operon. The gene on an inducible operon is always “off” with a repressor protein bound to it but can be turned “on” when a co-repressor binds to the protein causing a shape change that leads to the repressor protein falling off. Now, the RNA polymerase can bind to the promoter and start transcription. Our goal was to incorporate the plasmid into at least a few of the E. coli cells so that they can grow colonies large enough to see with our eyes and count their number.
Our hypothesis stated that if the pGLO plasmid is integrated into two colonies of E. Coli bacteria and two separate colonies of bacteria without the plasmid are allowed to incubate in separate LB agar plates, then the plates with transformed (+pGLO) bacteria will have the ability to grow in ampicillin and glow when introduced to arabinose while the untransformed plates will die when ampicillin is introduced because the pGLO plasmid contains the genes required for ampicillin resistance and GFP, which can be regulated in cells depending on the presence of arabinose, and E. Coli contains three enzymes that can break down arabinose, enabling the GFP to be active. Due to our findings and observations of the experiment, we are able to conclude that our hypothesis is correct. Using our background knowledge on plasmid integration, we knew that the E. coli that obtained it would be gaining new data to use. Learning that the pGLO plasmid contained ampicillin resistant, GFP, and araC, we developed the conjecture that whatever bacteria integrated this plasmid would be able to resist the antibiotic ampicillin and regulate GFP expression as an arabinose regulator protein. Thus, the hypothesis was created describing the capability of life with the pGLO plasmid when introduced into an environment with the death sentence of E. coli, otherwise known as ampicillin. Moreover, the addition of arabinose to the plate of LB agar and ampicillin would allow the bacteria to glow due to ara’s regulation of GFP, a green fluorescent protein. This is supported by the data and observations we collected throughout the experiment. For example, the plate with transformed bacteria in a plate of LB and ampicillin were able to develop into approximately 58 colonies. While there was not 100% coverage of the plate, like the plate of -pGLO with only LB in its plate, because not all the bacteria were able to take up the plasmid into its coding, as stated in our hypothesis. Therefore, the ampicillin resistant in the plasmid was able to combat the ampicillin in the bacteria’s environment, allowing for life and growth. In our +pGLO bacteria plate with LB, ampicillin, and arabinose, we saw approximately 18 colonies of green-glowing bacteria. This supports our thought that transformed bacteria will be able to glow due to the arabinose regulator protein combating the arabinose found in the agar plate by expressing GFP, along with the growth stated in the previous statement. We saw very distinct pictures between the plate with -pGLO bacteria and LB and the plate with -pGLO bacteria, LB, and ampicillin. To start, there was full coverage in the dish with only LB and -pGLO bacteria because there were only nutrients for the bacteria to use, with no antibiotic to affect these results in any way. In comparison, the plate that included ampicillin showed no growth due to how the bacteria had no plasmid with the coding for an enzyme to confer with the ampicillin in the plate. Using our data and observations sited, our group’s hypothesis was fully supported, and our objective for this experiment was met.
The objective of the pGLO Bacterial Transformation Lab, which was to successfully transfer a plasmid into a bacterial cell, was met and carried out successfully. Our data reflects this as there was growth on both the LB/AMP & +pGLO plate and the LB/AMP/ARA & +pGLO plate, which should not have occurred if the genes on the plasmid were not transcribed and carried out. Our group learned that the ARA gene in the plasmid actually is a gene that allows the bacterium with that gene to be able to metabolize the sugar arabinose that is the necessary co-repressor for the inducible operon GFP. The common misconception was that the ARA gene somehow produces the arabinose sugar but that was quickly disproved. Another thing that our group learned was that the purpose of heat shocking the bacterium was to create a gradient of temperatures. The gradient was formed by submerging the microtubule in ice water and then quickly transporting to the warm water, hence creating the gradient by having the insides of the bacterium be cold and their cellular wall warm. The reason that creating this gradient is because a draft pulling towards the center of the bacterium in created as a direct result of the temperature differences. The draft then pulls the plasmids into the bacterium and into their cytoplasm which was the objective of the experiment and procedure.
This lab connects to what we have learned in Chapter 20 as the chapter is all about Biotechnology. In this chapter, we saw the use of a plasmid to alter a bacterium. The steps to carry out this process was to remove a plasmid from an E. Coli bacterium, insert a segment or segments of DNA that contains the gene of interest (in our case the gene of interest was the ability to metabolize arabinose, ampicillin resistance, and the glowing jellyfish gene titled GFP), and then insert the recombinant DNA plasmid into a bacteria cell. The reason why researchers in the field may want to do this is so that they can give bacterium the ability to clean oil spills in the ocean or give plants pest resistance. Chapter 20 also taught us about using restriction enzymes to cut DNA so that we can isolate a gene of interest as well as insert it into the plasmid that has the right kind of opening for it to fit properly. By using the same enzyme to open the plasmid and cut the gene of interest, scientists can ensure a proper and snug fit. However, this lab not only refers to Chapter 20 but Chapter 18 as well. In Chapter 18 we learn of an inducible operon. An inducible operon is usually off but can be induced through a small molecule to turn transcription on. On the pGLO plasmid, the GFP gene is an inducible operon. Therefore, the reason that only the LB/AMP/ARA with +pGLO actually glowed in the dark was because the arabinose acted as the co-repressor to remove the repressor protein on the operator so the gene could be transcribed. As one can see in the data collected, the arabinose was necessary or else the bacteria colonies did not glow under ultraviolet light. This lab also slightly ties back into Chapter 17 as the chapter talks about the fact that there is only one genetic code for the DNA/RNA of all organisms which is why we can insert a certain gene of interest from one organism into another organism of a different species.
Throughout this experiment, our group can proudly say our lab was successful in both results and learning. Based on the data collected, our hypothesis rang true and aligned with each observation we made. Our group was able to see first-hand the myriad uses of biotechnology and plasmid integration at work. Instead of simply learning through the reading of Chapter 9 and/or previous chapters, we could visualize and physically grasp onto the knowledge through this experiment. Nevertheless, we can always make improvements to our experiment and skills in the lab for further times. For instance, the procedural aspect of this experiment could have been refined with a fine-tooth comb to allow us to get exact measurements, consistent timing, and, most importantly, the most accurate results. Not only can we alter the experiment for the better, we can also refine our lab skills. An example of improvement can be the delegation and smooth transitioning from one part of the lab to the other, such as the change from heat shock to further incubation. This can make our timing more precise, bettering the experiment as a whole. In this experiment, we observed how the better our skills are during the lab, the better the experiment went in general. In conclusion, by refining the experiment and out lab skills, our group can ensure improvement for the use of future times.
XII. Error Analysis/Improvements:
Although our lab was successful in the means of seeing the expected results, improvements can be settled to better enhance this experiment. To begin with, the measurements we took via micropipette were not consistent due to the number of air bubbles we saw when taking the measurement of the transformation solution (CaCl2). This disabled perfect viewing of solution before dispatching into the tubes. With this solution of cations, they are needed to complex with exposed phosphate heads of the phospholipid bilayer of the E. coli cell. With the instrumental error, the most drastic effect would be non exposure to the phospholipids, which could overall effect the transfer of plasmid into the cell.
In addition, the timing of incubation and heat shock were off by a few seconds. This is due to having to reset and adjust the timer when moving the tubes through the procedure. Overall, our group saw the expected results; however, these results could have been altered in a less impactful way. For example, we observed another group’s results and saw that they had larger cultures of bacteria and more of them. These errors could have altered our results in the sense of not giving the bacteria the proper treatment in order to see full potential for growth. In terms of transformation, the lack of proper incubation or heat shock will disable the neutralization of the phospholipids and create a gradient in which the DNA can pass through. Nevertheless, the outcome of our lab was minimally affected, as we saw the correct growth of death depending on the solution given. In conclusion, the errors made throughout the experimental process could have affected the overall results minimally, but we can come to the conclusion that small infractions to important steps can be impactful.