Sunday, December 18, 2022

A Long Goodbye To The Bloatware Frienemy


Student: John C Gibson

Engineering And Ethics

Fall 2022 Section 205

Professor: Christopher Phillips, Ph.D.

Word count: 1035

How Is Bloatware Different From Viruses?

A Long Goodbye To The Bloatware Frienemy 

I argue against giving bloatware, pre-installed software, a lifeline to survive in misery in a world that increasingly utilizes online applications that don’t need installation in the first place. I prescribe mercy killing the bloatware. A few trade names are omitted in this essay to avoid defamation.

Recently, particular desktop operating systems have included a self-upgrading campaign in the operating system's desktop screen that asks the user to consent to the upgrade to a newer version of the operating system. The campaign features tactics of a gray, obscured button for the “No” option and changing intervals of presenting the consent screen interlaced with antivirus software advertisements. An operating system upgrade historically can introduce incompatibility of applications, giving the operating system company’s applications a competitive edge. In the old days, OS companies paid hefty fines to settle suits with bloatware blatantly defying court orders with Internet browsers that were nearly impossible to uninstall. But recent advancements in intricate programming techniques can give any application more subtle tactics in coercing and deceiving the user into giving consent to different matters. Sophisticated programs implemented in contempt of court orders may be difficult to prove because the boundaries between coercion, deception, and marketing advertisements can be hard to locate inside an intricate web of parameters. 

The progressive trend of moral standards gives an ever-expanding inclusion of a circle of personhood and more rights to more subjects and objects, as reported in Vox's article (Samuel, 2019). A progressive view can personify or anthropomorphize desktop computers in different ways. It can be a good marketing strategy to promote a product by asking people to respect the product like a natural person with the right to survive, access to nutrients, and all needed resources as much as possible. After all, desktop computers are natural secretaries to their owners with calendars and daily planner apps. The sophisticated CPU scheduling system slows down CPU clocking during slower hours to save electricity, emulating the fluctuation of human cardiopulmonary performance throughout the day. And computer engineering can utilize anthropomorphization, too.

A modern desktop computer goes into hibernation and reboots at night after office hours to refresh its security upgrades, similar to human sleep, consolidating memory. It is not hard to reason why a manager in the desktop computer building business likes to promote the industry with the agenda of a well-upgraded, well-updated computer with a fresh and healthy mind to assist the owners, for example, at the kitchen counter with an interactive cookbook. The competitor of the kitchen counter Amazon Alexa has a humanoid voice; a desktop computer has a touchscreen allowing flipping book pages in stunning, life-like 3D renders, just like genuine human assistance. As a thought experiment, to achieve the stardom of Alexa, a desperate operating system company may coerce the user to consent to allow the operating system to instill a more famous humanoid voice than Alexa and exclude third-party text-speech software to prevent fragmentation. Bloatware can include a golf tournament show channel with the cookbook while the oven timer is ticking instead of Alexa’s voice-only functionalities. And the desktop computer may just become a great contender to the Alexa voice assistance appliance without the market knowing the stealth change. However, the online application trend diminishes the operating system’s role in a computer's functionality, and a voice-enabled, 3D multimedia assistant is just one click away within any browser. 

But the online-only application trend is not the only challenge bloatware and deceptive coercion face. The virtualization of desktop computer operating systems enables sharing of computers managed by expert desktop computer engineers. Increasingly, viruses need to get through the expert’s configuration of a desktop computer in fortified secure environments. The penetration rate of viruses might have declined with the virtualization trend. And experts can disable operating system self-upgrading campaigns that are nearly impossible to accomplish for average users. Expert configuration can happen in the virtual desktop setting or a physical, professionally managed setup.

Still, there can be traditional, retail desktop computers in certain circumstances. The work ethic of the managers is to be loyal to their employees and shareholders. Indoctrinating and educating consumers to build loyal customers for the parent company is a moral obligation. Education in schools often involves math techniques in ideal conditions, for example, calculating parabolic curves of throwing a ball without considering air resistance or a ball’s deflection with force. Education sometimes involves deception, putting pupils in an ivory tower or a religious atmosphere, and indoctrinating elite leaders’ national interests. And, in such a mindset, consumers need to be coerced and deceived. When an operating system company is fined an astronomical sum for contempt of court, the manager might receive a bonus for their loyal service. But are managers responsible for deception tactics enjoying their success and fame? Mac OS and Apple computers are relatively virus-free because Mac OS has a relatively small market share, and the virus economy depends on the sheer quantity of attacks to achieve penetration. When the penetration payoff of a virus is too low, the virus ceases to exist. When the market share of traditional, retail desktop computers shrinks, bloatware’s profit can become too low for the bloatware to be a viable business. And when businesses go out of business, layoffs follow.

But the average onlookers of the saga may not be so determined about letting bloatware die off. Sympathizers of the bloatware industry may argue that bloatware is very different from viruses. The bloatware industry can be a legitimate entry point job for novice engineers. The elite people of western societies have dominated all other people and natural resources for centuries, and progress toward equality and equity is slow. Why be so uptight about consent and giving autonomy? Keeping an open mind and allowing software bots and new engineers to enter our world can bring changes. But, the future of the virus or bloatware economy is uncertain if virtualization, browser-only appliance, and online applications continue expansion. If the managers of the bloatware business were new engineers we tried to save when they were younger, when the market trend goes down, it appears they will be victims who need sympathy to save again.

Works Cited

Samuel, Sigal. “Moral Circle Expansion: Should Animals, Plants, and Robots Have the Same Rights as Humans?” Vox, 4 Apr. 2019,

Sunday, October 16, 2022


 Word count: 341 


Located in Washington, DC, National Transportation Safety Board is an independent agency directly under the federal executive branch. It was split from the Department of Transportation in 1975 with the federal Independent Safety Board Act (Congress, 1994). The current chairwoman is Jennifer Homendy, appointed by former president Donald Trump. Independent agencies outside cabinet departments and directly under the president include the CIA and FCC. Understandably, most federal independent agencies established themselves close to Washington, DC. The Potomac River runs along the west side of Capital Hill, so the agencies generally cluster on the river shore neighborhoods. L'Enfant Plaza houses NTSB’s headquarters on the east shore. On the west shore, the Langley headquarters of the CIA is about 6 miles west of NTSB. Lincoln Memorial is within walking distance from L'Enfant Plaza. However, for more recreation, the nearby National Harbor is an area with waterfront dining, a shopping mall, and a Ferris wheel, about 5 miles south of NTSB. 

The general feature of the 6 miles west of NTSB is a dense highway system with substantial maintenance. Amazon has a large data center built in the area as part of the communication artery (Sverdlik, 2015). The buildings for the vital data center facilities are tall and imposing. The federal workers in Washington, DC, may have long careers in the area. The former NTSB aviation safety director Thomas Haueter has a career spanning 28 years with NTSB, gaining expertise and experience (Adair, 2004).

The laboratory of NTSB headquarters has voice tape players, flight instrument decoding software, and other black box testing tools (Adair, 2004, p38). However, specialized testing tools are only available in manufacturers’ labs, which include engine and heavy machine testing tools. The airworthiness investigator has broad authority and legislated power to acquire manufacturers' necessary assistance and equipment. The “goto” team’s workplace is often the hotel rooms with extra phone lines (Adair, 2004, p28). In investigating USAir flight 427’s crash, Thomas Haueter boarded over a dozen flights with the Boeing 737-3 series to acquaint himself with the 737 models (Adair, 2004)

Works Cited

"H.R.2440 - 103rd Congress (1993-1994): Independent Safety Board Act Amendments of 1994.", Library of Congress, 25 October 1994,

Sverdlik, Yevgeniy. “Fire at Amazon Data Center Construction Site in Ashburn Contained.” DataCenter Knowledge, 9 Jan. 2015,

Adair, Bill. The Mystery of Flight 427: Inside a Crash Investigation. Illustrated, Smithsonian Books, 2004.

Thursday, June 30, 2022

 Viking Study Essay A Very, Very Small Progress

 A Very, Very Small Progress

Excerpt from the Heimskringla Saga - "The citizens had plenty of food and whatever else they needed to withstand a siege. So Harald hit on a plan. He had his fowlers catch some little birds which nested in the city and flew out to the forest during the day to find food. Then he had chips of resinous wood, smeared with wax and brimstone, attached to the birds’ backs and set alight. The instant the birds were released, they all flew straight back to the city to seek out their young and their nests in the thatched roofs. The fire then spread from the birds to the house-thatches, which were made of reeds or straw. Even though each bird carried only a small spark of fire, there was soon a huge conflagration as lots of birds brought fire to thatches throughout the city. One house after another burst into flames until the whole city was ablaze.

The inhabitants abandoned the city and begged for mercy, even the ones who had often spoken haughtily and dismissively about the Greek army and its leaders. Harald spared everyone who asked for mercy and took control of the city. …" (McDonald 297).

It is intriguing to see a modern-like treatment of the enemy by vikings when no one is explicitly depicted as injured or killed in a siege, only, maybe, implied. That is not very common for a viking saga. The writer is the friendly Icelandic diplomat Snorri Sturlusson to Norway at the turn of the twelfth century. Harald refers to Harald The Hard Ruler, a future viking king of Norway at the time of the action. No one is harmed by the constraint of resources because “plenty of food and whatever else '' are available during the siege in the viking king’s early military training career (McDonald 297). The writer receives a substantial “gift” from the Norwegian royal family and discloses the gift (Byock 15). He openly declares that another saga, Lay of Ragnar, is dedicated to “honor” the gifting royal family (Byock 15). 

The besieged city in question is in Sicily, which is one thousand and five hundred miles southeast of Norway, and the year is 1038. For a diplomat to compose a summary report, Snorri succeeds in negotiating and navigating between pros and cons of accurate descriptions and empathetic condolences to reconcile all parties involved, when “asked for mercy” and “spared” are said, instead of words like “granted” by the trainee king, a prince by definition (McDonald 297). For this particular saga, the audience includes the royals of the very characters of the saga, the nobles that served the royals, and the ruled commoners. The readership is diverse, and it is a matter of checks and balances between Snorri’s light-hearted words, such as “whatever else”, on his mind and the serious, business side, work product.

In the first sentence, the siege is set in motion by the Byzantine Empire’s effort to consolidate power, to establish a hierarchical society, where piety is paramount. It has been at least five hundred years since the fall of the empire-sized Rome government to the story’s action time. Ever since the fall of Rome, the isolated, small, and unstable societies treat each other “dismissively” (McDonald 297). And countries dismissing one another can lead to wars.

From the second to the fifth sentence, as promotional material for the royal family, the military skills of The Hard Ruler appear godly resourceful, defying calculation of power of resources when local “resinous wood” is utilized as zero-cost weapon, and “little birds'' become projectors with perfect range and precision when “they all flew straight back to the city” (McDonald 297). Snorri Sturlusson’s diplomatic mission must have been successful for the hosting country to gift him. With his new generation’s novel prose, vernacular form of writing, he can speak his mind directly, not very different from twenty-first century science paper writing. 

People know that sagas often hint on the outlines of historical facts, and outright trivialization can add insults to injury. So near the ending sentence, the politically correct way is said that the “Greek army” wins their battle (McDonald 297). Norse or Rus is not named as the army. There are a few options for the name of the “Greek army” (McDonald 297). According to modern research, the army in the saga is actually a joint force of the Varangian nordic mercenaries to Byzantine and local Greek soldiers, and the mercenaries either trade goods for silver as a side job or straight out rob silver (Haywood 109). Foreign legionnaires is the modern term for mercenaries. Legionnaires navigate through their ranks in a foreign country, negotiating between personal sacrifices and meager gains on a daily basis. And the young Harald is therefore a legionnaire in such a situation when “silver mines became exhausted” “around 965” (Haywood 108). 

Specifically, the Heimskringla saga is in the genre of kings saga, which are meant to be glorious. The Hard Ruler is a very ambitious king bent on conquering England to form a united Norway-England country. His plan is well-known by historians. But with the ambitious king recently deceased, the Heimskringla may appear as an obituary in some readers’ minds, albeit a mixed-emotion one, with an “ablaze” motif of norse funeral cremation in the excerpt (McDonald 297). However, in the roller coaster of emotions, the lesson of history is still preserved. The exact actions and details are not likely accurate in the saga. But it is said that the Heimskringla saga provides “valuable insight of Scandinavian mercenaries in the East” (McDonald 297). 

Reading the whole passage, critical readers can see the tell-tale signs of progress, the smart military tactics at play in the writing. Berserkers are not mentioned in the 1038 siege. Instead, the traditional hit-and-run viking tactics morph into something more modern-like with “took control of the city” instead of taking booties of the city (McDonald 297). But modern tactics require resource management, logistics that can not defy laws of physics or mathematical calculations on economy. When vikings become modern-like, centralized forces, resources can not be stretched indefinitely. In the decade of laying the siege, Scandinavians have both eastward Swedish Kievan Rus integration and westward migration to escape well known forced religious conversion, a religious persecution, with new “tax” schemes to slow down the human resource drain (Haywood 108) (McDonald 312). It does not appear sustainable for the Scandinavian power, as a whole, to expand eastward and westward simultaneously, let alone Norway expanding on both spheres by itself. Norway does not have enough resources left to take over England in the 1066 monarch vacuum in the west after resource drain, both human and economical. As a result of the shortcoming, Norway suffers heavy losses in the war for the English throne in 1066 when the story’s character, the beloved young king, is killed abroad. The supposed united Norway-England country becomes a lost cause, marking the end of the viking age by modern view.

But progress is made nonetheless. “Heimskringla” translates to “The Circle Of The World” (McDonald 297). And the unintended value of the saga is not limited to the “insight ” of mercenary life of the era (McDonald 297). It gives concerned learners in the future a reality check about the consequences of social unrest and religious persecution. It shows the consequence of intolerance. It contributes to understanding the year 1066. It shows that the outcome of the war could have been different with understandable change. 

But progress, however small, is progress.

Vafthrudnir 2022

Works Cited

McDonald, R. Andrew, and Angus A. Somerville. The Viking Age: A Reader. University of Toronto Press, 2020. 

Byock, Jesse L. The Saga of the Volsungs: The Norse Epic of Sigurd the Dragon Slayer, Penguin Books, London, 2012, pp. 15–15.

Haywood, John. The Penguin Historical Atlas of the Vikings. Penguin Books, 1995. 

Monday, May 2, 2022

Micro-scale pAMP/KAN Recombinant Plasmid Production

 John C Gibson, Hunter Lyons

University Of Massachusetts, Lowell

Biological Sciences, Experimental Methods

Section 802A, Spring 2022

Micro-scale pAMP/KAN Recombinant Plasmid Production


pAMP, short for plasmid DNA with a gene for ampicillin resistance, and pKAN, short for plasmid with a gene for kanamycin resistance, were cleaved by commercial grade mixture of BamHI(the first identified restriction enzyme from the H strain of Bacillus amyloliquefaciens) and HindIII(the third identified restriction enzyme from the d strain of Haemophilus influenzae) in this study. The cleaved fragments were ligated to produce recombinant plasmid DNA of pAMP/KAN. The dual antibiotic resistance efficacy with this novel plasmid for E. coli bacteria was tested successfully. The simple recombinant plasmid DNA was cloned and purified. Verification measures were performed in each step of digestion, ligation, and purification.

Introduction and Background

Recombinant DNA technology underwent significant growth since the 1970s with biological research and applications, such as the first isolation and cloning of mammalian β-globin “gene of interest” by Prof. Philip Leder at Harvard University[1]. The cloning techniques improved over time, such as the change from Ethidium Bromide with UV light in electrophoresis, used from the 1970s[7], to newer fluorescence agents and alternative light sources. The goal of this study was to establish a procedure utilizing current commercial supplies to produce small quantities of recombinant DNA and to test the product. This study aimed to give an update of materials and procedures appropriate for 2022. The cloned genes on plasmid products had very broad applications, including researchers establishing therapy of cardiovascular diseases[16] based on recombinant plasmids. The importance of understanding and grasping current material supply trends of this subject could not be overstated. 

Gene of interest, as mentioned, was essentially the gene studied with its protein originated from its transcribed mRNA from DNA and tested on life cells to determine the gene’s properties. Molecular cloning was the needed procedure to selectively bring the needed DNA, and subsequently its derived mRNA, to a proper quantity/dosage for experiments. Recombinant technology gave the bacterial clone growth selection advantage for the gene of interest, either by the recombinant plasmid carrier’s own selection advantage or by the payload gene’s. In this study, the KANr was tested for its antibiotic resistance property and therefore studied as a gene of interest that needed to be selected and cloned. pAMP was considered the carrier plasmid in this study because it provided the origin of replication for cloning. KAN lacked the origin of replication. It was hypothesized that simple recombinant pAMP/KAN was more easily produced than superplasmid or double transformation.

Overall, the cloning started with BamHI and HindIII enzyme digestion on stock plasmids because it was well known that this enzyme mixture preserved the KANr gene during cleavage of the carrier plasmid and the payload plasmid. In this study, SybrSafe was used as the fluorescence agent with blue light to verify cleavage progress and to verify the final product. The term, one-pot[9] ligation, could be perceived as inadequate precision by etymology, but the efficiency of the procedure made it suitable for this study to ligate the cleaved DNA fragments. In this study, a commercial grade, competent E. coli strain was used for transformation to incorporate the gene of interest into the living cells for cloning. And after the transformation, the inoculation loop was utilized for single-colony identical genome selection. Finally, the mini preparation procedure followed a textbook example to purify the plasmid DNA product samples.

Materials and Methods

Plasmid DNA Cleavage

BamHI and HindIII restriction enzyme mixture were utilized in this step, identical to textbook procedure[10], to cleave 2 plasmid DNA molecules, namely pAMP and pKAN. 5.5μL of pAMP stock plasmid and 5.5μL of pKAN stock plasmid were digested in 2 separate reaction tubes. The enzyme mixture was supplied by New England Biolabs(NEB for short), and stock plasmids were supplied by Carolina Biological Supply. The concentration of the stock plasmid DNAs were 0.2μg per 1μL. For each reaction tube, 2μL of BamHI/HindIII enzyme mixture was used. The reactants were suspended in a 1X restriction buffer, each tube 15μL by volume. Both digestions were then incubated at 37°C for 89 minutes. However, after the initial 30 minutes in incubation, a 5μL sample from each reaction tube was removed for electrophoresis analysis to verify digestion progress. The remaining 10μL sample of each reaction tube completed the full 89-minute incubation.

Gel Electrophoresis Of Digested Fragments

Following the textbook procedure[11], to verify digestion progress, a 5μL sample, drawn from each reaction tube at 30 minutes into the digestion, underwent electrophoresis in a 0.8% agarose gel and utilized SybrSafe as the fluorescence agent. The SybrSafe fluorescence agent was used to reduce toxicity of waste products. 1X TBE(Tris/Borate/EDTA) was used as the gel base as well as the electrophoresis solution[10]. 1μL loading dye was utilized as well as a standard λDNA HindIII digestion loaded for simultaneous electrophoresis. Electrophoresis was performed at 130 volt for 33 minutes. Transillumination was performed on the resulting gel with blue light.

One-Pot Ligation

The digestion enzymes in the digestion tubes were first heat-inactivated[12] by a 10-minute incubation of the digestion tubes at 79°C(slightly higher than textbook[12] inactivation temperature) after the 89-minute digestion. Then a 3μL sample from each of the completed digestions was drawn and combined in a single reaction tube to undergo recombination. The reactants were suspended in a 1X ligation buffer with 1μL of T4 ligase to form a 20μL total reaction volume. The ligation was incubated at 20°C for 18 hours.

Bacterial Transformation With Recombinant Plasmid

After completing ligation, 10μL of ligation volume was mixed into 100μL of commercial grade competent Douglas Hanahan strain(DH5α for short) of E. coli suspension to transform the bacteria. A heat-shock was administered at 42°C bath for 30 seconds followed by immediate cooling in an ice bath for 30 minutes, then the transformation suspension was mixed into 900μL of Super Optimal Broth With Catabolite Repression media(SOC media for short). The media with DH5α content was then placed in a shaking incubation chamber of 37°C at 250 RPM for recovery for 1 hour.  

Bacteria Plate Culture For Gene Selection

At the end of the recovery time, the recovery media with transformed DH5α were plated  onto 4 petri dishes, the first plate with only lysogeny broth in agar form(LB agar for short), the second plate with LB agar with added ampicillin, the third plate with LB agar with added kanamycin, and the fourth plate with LB agar with both ampicillin and kanamycin added, per textbook procedure[13]. Each plate received 230μL(larger volume than textbook procedure) of the recovery media with transformed DH5α content, and sterile spreaders were utilized to evenly spread the bacterial media across the entire agar surface. The 4 selection culture plates were incubated at 37°C for 18 hours.

Bacteria Selection And Gene Cloning

After 18 hours of growth, two isolated colonies in the plate containing LB agar with both ampicillin and kanamycin were picked up by sterile inoculation loops for identical genomes. Each identical-genome colony inoculated 5mL of cloning media in a separate culturing tube. Liquid LB with both ampicillin and kanamycin was used as the cloning media. The growth media was incubated at 37°C for 18 hours.

Plasmid DNA purification mini preparation

After 18 hours of cloning in the liquid media, the cloned DH5α E. coli cells were condensed into a pellet by centrifugation at 1400 RPM for 1 minute, per textbook procedure[14]. Each cloning media’s cell strain on its own sample tube in this step. Excess liquid drained. Then the cell pellet was loosened up with ice-cold 100μL GTE(glucose/Tris/EDTA mixture) solution and then lysed with 200μL SDS/NaOH(a mixture of SDS detergent and NaOH) to release plasmid DNA molecules from cell envelopes. The alkaline lysis was given 5 minutes to complete, then 150μL of ice-cold acidic potassium acetate KOAc was added to condense cell debris and to bring the pH level of the suspension back to neutral. A further wait time of 5 minutes was given, then condensed cell debris was precipitated out by centrifugation at 1400 RPM for 5 minutes and discarded. 

The suspended plasmid DNA from the supernatant was treated with added isopropanol alcohol, 400μL for each strain of sample, stood at 18°C for 2 minute, and then immediately underwent centrifugation at 1400 RPM at 0°C for 5 minutes. The 5-minutes centrifugation precipitated the DNA sample at the bottom of the reaction tube, which was further resuspended with added pure ethanol, 200μL for each sample. The DNA sample was further re-condensed with centrifugation at 1400 RPM for 3 minutes, and excess ethanol drained. The DNA sample was finally allowed to dry for 10 minutes, and then re-suspended in 15μL of TE(Tris/EDTA).

Plasmid DNA product verification - BamHI and HindIII cleaving

5μL of each of the 2 DNA samples were drawn and mixed into BamHI and HindIII restriction enzyme mixture in this step, per textbook procedure[15]. For each of the 2 digestion tubes and the control group of 5μL factory pAMP/pKAN mixture, 2μL of BamHI/HindIII enzyme mixture was added. All 3 digestion tubes and 2 non-digestion tubes of 5μL of each of the 2 DNA samples were suspended in a 1X restriction/RNase buffer, each tube filled with distilled water to reach 10μL by volume. All 5 tubes were then incubated at 37°C for 43 minutes. 

Plasmid DNA product verification - Electrophoresis

All 5 samples underwent electrophoresis in a 0.8% agarose gel and utilized SybrSafe as the fluorescence agent. 1X TBE was again used as the gel base as well as the electrophoresis solution. 1μL loading dye was utilized again, as well as loading the standard λ DNA HindIII on a separate lane for simultaneous electrophoresis. Electrophoresis was performed at 130 volt for 33 minutes. Transillumination was performed on the resulting gel with blue light.


With the DNA digestion procedure on the commercially sourced pAMP and pKAN plasmids, the electrophoresis of the cleaved fragments in Figure 1(A) showed multiple bands of cleaved DNA in both experiment lanes of pAMP and pKAN of plasmids. The NEB lane contained all the 7 well-known λ(a bacteriophage) DNA fragments, among which, 4361 base pairs(bp for short), 2322 bp, 2027 bp, and 564 bp.

“pAMP” lane contained 2 bands and 1 smear. Using distance estimation, the first band in between the 4,361bp and 2,322bp markers was estimated to be 3,755bp, the second band close to the 564bp marker was estimated to be 784bp. There was a smear between the 4,361bp and 3,755bp. 

“pKAN” lane also contained 2 bands, the first band just slightly higher than the 2,322bp marker, estimated to be 2,332bp, and the second band just slightly lower than the 2,027bp marker, estimated to be 1,861bp.

Figure 1(B) showed multiple bands of cleaved DNA fragments in both experiment lanes of pAMP and pKAN of plasmids by the instructor. The instructor’s ideal gel’s NEB lane in Figure 1(B) also had 7 bands. Also in the instructor's ideal gel, both the pAMP lane and pKAN lane contained 2 clear bands. The 2 bands in pAMP lane were 3,755bp and 784bp. The 2 bands in pKAN lane were 2,332bp and 1,861bp. The instructor’s ideal gel’s pAMP and pKAN lanes did not have smears.

The remaining digestion, not used in the initial 30 minute digestion electrophoresis analysis, underwent ligation/recombination and transformed DH5α E. coli cells with recombinant plasmids in the bacterial transformation sub-procedure. The transformed E. coli colonies were shown in Figure 2(A)’s plating result, which displayed growth by media nutrients in all plates. Figure 2(B) summarized the relative growth per plate with their experimental control category.  

As shown in Figure 2(A) photograph, the LB Only plate, as the positive control group as indicated in Figure 2(B), had the most vigorous white lawn of growth of DH5α E. coli bacteria. The LB+AMP agar plate for ampicillin-only inhibition control had slight dotted white growth with approximately 40 colonies. The LB+KAN plate for kanamycin-only inhibition control also had slight dotted growth with approximately 40 colonies. The LB+AMP+KAN experiment group had the least growth, approximately 7 colonies of small white dots.

Two single colonies from the LB+KAN+KAN plate, after liquid culturing, cloning and undergoing DNA purification, produced the electrophoresis bands in the following Figure 3(a).

Figure 3 showed this experiment’s result of electrophoresis side-by-side with instructor ideal electrophoresis of simple-recombinant pAMP/KAN results. The SMP1- and SMP2- lanes in Figure 3(A) of this study experiment had a single band between 6557bp and 4361bp, estimated to be 5616bps. The SMP1- and SMP2- lanes in Figure 3(B) of the instructor ideal electrophoresis had a smear between 6557bp and 3755bp, estimated to be 5616bps by supercoil that had heavier molecular weight than the apparent travel distance. 

The SMP1+ and SMP2+ lanes in Figure 3(A) of this study experiment had 2 bands, one between 4361bp and 2322bp markers, estimated to be 3755bp, and another band slightly lower than the 2027bp marker, estimated to be 1861bp. The SMP1+ and SMP2+ lanes in Figure 3(B) of the instructor’s ideal electrophoresis also had 2 bands, one between 4361bp and 2322bp markers, estimated to be 3755bp, and another band slightly lower than the 2027bp marker, estimated to be 1861bp. Figure 3(A)’s pAK+ lane had 3 bands, estimated to be 3755bp, 2332bp, and 1861bp. Figure 3(B)’s pAK+ lane had 4 bands, estimated to be 3755bp, 2332bp, 1861bp, and 784bp.

Discussion And Analysis

The overall goal of this study could be restated as cleaving stock pAMP and pKAN as completely as possible to ligate them to create as much novel recombinants containing  both AMPr and KANr as possible. The 30-minute digestion analysis shown in Figure 1(A)’s pAMP lane had 2 bright bands of well known sizes, 3755bps[4] and 784bps[4], indicating intense digestion progress, which was desired for achieving the overall goal. pKAN lane also showed 2 bright bands of well known sizes, 2,332bp[4]s and 1,861bps[4] respectively, indicating intense digestion progress as well. The band locations at 3755bps, 784bps, 2332bps, and 1861bps made sense because typical Bam and Hin group restriction endonucleases cut DNA molecules on average once every 4000 base pairs[5][6]. The combined BamHI/HindIII therefore cut average 2000 bps fragments as expected. The fact that this experiment’s band patterns matched the instructor’s ideal gel band patterns of Figure 1(B), namely 3755bps and 784bps in pAMP lane and 2,332bps and 1,861bps in pKAN lane, reaffirmed the success of enzyme digestion. 

Furthermore, figure 1(A)’s pKAN lane indicated that digestion was complete within 30 minutes by the total absence of the original 4193bp plasmid molecules between the 23,130bps marker and the estimated 2332bps distance, which was identical to instructor ideal gel’s pKAN lane of Figure 1(B). Figure 1(A)’s pAMP lane’s smear near the 4361bps mark implicated incomplete digestion at the 30 minute time. The undigested 4539bp molecules traveled slightly further than the 4361bps mark by supercoiling, which reduced the molecule’s apparent size. However, the smear of the 4539bps molecules was relatively faint compared to the digested bands of 3755bp and 784bp. This contrast meant that, by the end of 89 minutes of the full digestion period, the pAMP digestion should have also been complete. 

The results of all 4 culture plates in Figure 2(A) all made sense because the pAMP and pKAN digestion was confirmed to be successful by electrophoresis, so the ligated fragments must have transformed DH5α to give rise to antibiotic resistance in LB+AMP and LB+KAN and LB+AMP+KAN plates. For the LB Only plate, the vigorous growth in this control group meant that LB medium was fit for the experiment, and that the severely inhibited growth in the LB+AMP+KAN experiment group was due to antibiotic inhibition, not due to poor nutrient supplies. The partially inhibited growth in the LB+AMP plate meant that the experiment’s ampicillin was effective, and hence the severely inhibited growth in the LB+AMP+KAN was not the result of kanamycin bactericidal action alone. The partially inhibited growth in the LB+KAN plate meant that the kanamycin was effective, and hence the severely inhibited growth in the LB+AMP+KAN was not the result of ampicillin’s inhibition action alone. The 3 control groups together showed that the LB+AMP+KAN plate exerted high selection pressure favoring cells with both AMPr and KANr genes, and that the isolated colonies in the LB+AMP+KAN plate must had both AMPr and KANr genes in each cell. It was not reasonable to attribute the dual antibiotic resistance to either a single transformation of AMPr or KANr in any individual cells because both ampicillin and kanamycin were shown to be effective and fit for experiment by the 2 mutually assured control groups of LB+AMP and LB+KAN. The growth in the LB+AMP+KAN plate thus unquestionably indicated the success of plasmid transformation of both  AMPr and KANr genes into individual cells, as expected.

Considering that the efficiency of plasmid transformation of bacteria decreases with the increase in plasmid size[8], the pAMP/KAN novel plasmid of 5616bps (3755bps and 1861bps combined) was more likely to enter the E. coli cell than the superplasmid pAMP/pKAN of 8732 bps (4593bps and 4193bps combined). Comparing the chance of a single pAMP/KAN novel recombinant DNA plasmid entering the cell to the chance of dual plasmids of both the regenerated pAMP and pKAN entering the cell, the single event of pAMP/KAN entering the cell was more likely. The resulting electrophoresis pictures in Figure 3(A) demonstrated such expected likely results by the total absence of superplasmid of 8732bp band in any lane and the total absence of 2332bp band in the SMP1+ and SMP2+ lanes. In Figure 3(A) the simple recombinant of 5616bps appeared as a clear band in SMP1- lane between the 6557bp and 4361bp markers, which made sense by the simple ordering of the molecular sizes. If double transformation had occurred, the cloned pKAN would have been digested as 2332bp and 1861bp bands. The absence of the 2332bp band in the SMP1+ and SMP2+ lanes rejected the interpretation of double transformation giving the dual-antibiotic resistance seen in Figure 2(A)’s LAB+AMP+KAN plate.

The same band of 5616bp in SMP1+ lane appeared fainter in the SMP2- lane, but the appearance did not alter the inferred molecular size by the band travel distance. The fact that the instructor’s ideal gel band pattern in Figure 3(B) being identical to this experiment’s Figure 3(A) meant that simple recombination ligation unmistakably occurred in this study experiment.


The goal of this study was to establish a procedure to cleave the factory stock pAMP and pKAN as completely as possible to produce as much recombinant plasmid DNA with AMPr and KANr genes together in plasmid in a living cell for cloning to a micro scale. And as shown in discussion, this study experiment successfully produced the simple recombinant pAMP/KAN plasmid , which was 5616bps in length, 3755bps(pAMP) and 1861bps(KAN) combined. Overall, the goal of this study was achieved. The micro scale production steps in the experiment followed the well known procedures as much as possible and the desirable results were achieved. The cleavage of the stock factory plasmids was considered complete, the one-pot ligation created the needed recombinant, and the inoculation loop selection of bacteria picked out the needed simple recombinant colonies. The improvements in the future could be the loading for electrophoresis. More practice would result in more complete DNA material loading in the loading well and brighter electrophoresis bands.   


[1] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 143.

[2] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 495.

[4] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 451.

[5] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 111.

[6] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 144.

[7] Aaij, C, and P Borst. "The gel electrophoresis of DNA." Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, vol. 269, no. 2, 31 Dec. 1971, pp. 192-200.

[8] Szostková, Monika, and Dana Horáková. "The effect of plasmid DNA sizes and other factors on electrotransformation of Escherichia coli JM109." Bioelectrochemistry and Bioenergetics, vol. 47, no. 2, Dec. 1998, pp. 319-23.

[9] Potapov, Vladimir, et al. "Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly." ACS Synthetic Biology, vol. 7, no. 11, 2018, pp. 2665-74.

[10] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 443-450.

[11] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 449.

[12] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 453-454.

[13] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 463-467.

[14] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 427-429.

[15] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 489-491.

[16]Williams, Paul D. Williams D., and Paul A. Kingston A. Kingston. "Plasmid-mediated gene therapy for cardiovascular disease." Cardiovascular Research, vol. 91, no. 4, 1 Sept. 2011, pp. 565-76.