Daily Archives: July 9, 2024

Step 1Obtaining the mutant insert by error-prone PCR and the control insert

The DsRed2 gene was isolated using the plasmid pDsRed2 (Clontech, Cat. No. 632404, UniProt Q9U6Y8) as a template (Fig.1A). Error-prone PCR of the DsRed2 gene was performed using the GeneMorph II Random Mutagenesis kit, following the manufacturers protocol The primers DsRed2-EcoRI-F and DsRed2-BamHI-R (Table 1) were used and the PCR conditions included one cycle at 94C for 2min, followed by 30 cycles at 94C for 15s, 68C for 30s, and 72C for 60s, with a final elongation step at 72C for 5min. The products of error-prone PCR are referred to as the mutant insert in this text. The DsRed2 gene without mutations (Fig.1A) was amplified using the same primers and high-fidelity polymerase (TAKARA LA Taq DNA, Clontech Cat. No. RR002A) as a control for the procedure (referred to as the control insert). The PCR conditions for this were 94C for 2min, followed by 30 cycles of 94C for 15s, 60C for 30s, 72C for 2min, and a final cycle at 72C for 5min. After PCR, the amplicons were verified on 1% agarose gel electrophoresis and purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare).

Graphic representation of the main methodological steps for comparing the Ligation-Dependent Process Cloning method (LDPC) and Circular Polymerase Extension Cloning (CPEC). (A) Step 1 Obtaining the Mutant Insert by Error-Prone PCR and the Control Insert. The DsRed2 gene was isolated from the plasmid pDsRed2 through error-prone PCR using specific primers and conditions, resulting in the mutant insert. The control insert was also isolated from the plasmid pDsRed2, but a high-fidelity polymerase was used. (B) Step 2Ligation-dependent process cloning. A vector was prepared by cleaving the pDsRed2 plasmid with BamHI-HF and EcoRI-HF enzymes, followed by digestion of all fragments (control insert and mutant insert from Step 1) using the same restriction enzymes, and ligation reactions were performed using T7 ligase. (C) Step 3Circular Polymerase Extension Cloning CPEC. The mutant insert, along with the control, was amplified via PCR, quantified, and cloned into the pCDF1b (GenBank Accession Number OR900361.1) expression vector using CPEC with overlapping primers.

Initially, the pDsRed2 plasmid was cleaved (Fig.1B) using the enzymes BamHI-HF (New England Biolabs, Cat. No. R3136) and EcoRI-HF (New England Biolabs, Cat. No. R3101) to get the vector. Afterwards, BamHI-HF and EcoRI-HF restriction enzymes were used to digest all fragments (control insert and mutant insert Step 1). This digestion took place over an incubation time of 2h at a temperature of 37C. The enzymes were inactivated for 20min at 65C. Digested fragments were quantified on the Qubit fluorimeter (Life Technologies, Brazil) using the Quant-iT dsDNA BR Assay kit (Invitrogen, Brazil). A 1:1 ratio was used for the ligation reactions. The vector (pDsRed2) was at a concentration of 81.7ng/L and the inserts were at a concentration of 84.1ng/L. The ligation using the T7 ligase (New Englands, Biolabs Cat. No M0318) was carried out according to the manufacturers protocol and was conducted in triplicate.

A total of the 1 L of product from each ligation was transformed into 40 L of electrocompetent Escherichia coli TOP 10 bacteria (0.2cm cuvette, 2.5kV/cm, 25 F, 200 , 1 pulse) using the Gene Pulser Xcell electroporation system (BioRad). The cells were grown in 480 L of SOC medium (2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5mM KCl, 20mM glucose) for 1:30h at 37C with constant shaking at 243g in a Stuart Shaking incubator SI500 orbital shaker (Stuart, Brazil). After incubation, the inoculants were seeded in plates containing Luria Bertani (LB) agar medium and antibiotic spectinomycin (100g/mL) and incubated for 16h at 37C. The bacteria transformed with the product of each ligation were screened for strong fluorescence using the Safe Imager 2.0 Blue Light Transilluminator (Invitrogen) with excitation at 470nm. The plates obtained were photographed and the total number of colonies on each plate was determined. The plates for the controls and mutants were quantified using microscopy and counted with ImageJ software.

We utilized the construct that was obtained and chosen from Step 2 (pDsRedmut) as a template for the construct that included the mutant insert. PCR reaction was performed using the primers Mut/Dsred2-F and Mut/Dsred2-R (Table 1) and the TAKARA LA taq high fidelity DNA polymerase (5U/L TAKARA LA Taq, 10X LA PCR buffer II (Mg2+ free, 25mM MgCl2, 0.25mM dNTP). The PCR conditions were one cycle of 94C for 2min (initial denaturation) followed by 30 cycles of 94C for 15s, 66C for 30s, and 68C for 3min, and a final elongation of 72C for 10min. After PCR, the fragments (hereafter mutant) were quantified using the Quant-iT dsDNA HS Assay kit (Invitrogen, Brasil). The same procedure was done in the DsRed2 gene as a control. The mutant gene and the control were cloned into the pCDF1b expression vector (Novagen, Cat. No. 71330-3) (Fig.1C). The ligation of fragments (DsRed2 and DsRed mut) with vector (pCDF1b) was done via CPEC with the primers PCDF-F and PCDF-R (Table 1). These oligonucleotides have an overlapping sequence (bases under-arrayed in the sequence) with the product mutant for CPEC to occur.

The PCR for CPEC was carried out using the TAKARA LA Taq enzyme (Clontech Cat. No. RR002A), following the conditions: 94C/2min, 30 cycles of 94C/15s, 63C/30s, 68C/4min and 1 final cycle 72C/5min. The template DNA for the CPEC reaction was the double-stranded fragments of the mutant and the vector pCDF1b was added in a 1:1 ratio. In the first PCR cycle, the fragments are denatured. In the following cycles, the single strands are ringed in the sequence in which they overlap, and it is from this overlap that the fragments extend to form the double strand of the circular plasmid pCDF1b-DsRed2mut and pCDF1b-DsRed2, respectively. The fragments were analyzed using 1% agarose gel electrophoresis.

The expression vectors produced (pCDF1b-Mutant and pCDF1b-DsRed2) were transformed into electrocompetent Escherichia coli BL21-DE3 by electroporation (0.2cm cuvette, 2.5kV/cm, 25 F, 200 , 1 pulse) using the Gene Pulser Xcell electroporation system (BioRad). The transformed bacteria were seeded in plates containing Luria Bertani (LB) agar medium and antibiotic spectinomycin (100g/mL) and incubated for 16h at 37C. After transformation, bacterial colonies were inoculated into liquid Luria Bertani (LB) medium, using antibiotic spectinomycin (100g/mL) as a selective agent, incubated for 16h at 37C with constant shaking at 243g in a Stuart Shaking incubator SI500 (Stuart, Brazil). Subsequently, the plasmids were purified using the Ilustra- Plasmid Prep Mini Spin Kit (GE Healthcare). The plates obtained were photographed and the total number of colonies on each plate was determined. The plates for the controls and mutants were quantified using microscopy and counted with ImageJ software.

The selected bacterial colonies were inoculated into liquid Luria Bertani (LB) medium, using the antibiotic spectinomycin (100g/mL) as a selective agent, incubated for 16h at 37C with constant shaking at 243g in a Stuart Shaking incubator SI500 orbital shaker (Stuart, Brazil). Subsequently, the plasmids with mutant and control inserts were purified using the Ilustra- Plasmid Prep Mini Spin Kit (GE Healthcare). After purification, the plasmids were sequenced using the oligonucleotides PCDFBGL-Seq-F and PCDFBGL-Seq-R (Table 1) to confirm binding using the CPEC and LDCP methodologies.

One-way ANOVA was used for the statistical analysis of the data, with a significance threshold of p<0.05. To make sure the test assumptions were met, tests for homogeneity of variances and residuals normality were performed before to the ANOVA. Specifically, Levenes test was used to assess the homogeneity of variances, and the ShapiroWilk test was employed to evaluate the normality of residuals.

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Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries -...

QUICK FACTS

Name: "Immortal" seagrass meadow

Location: Shark Bay, Western Australia

Coordinates: -25.8829486505056, 113.90591540490992

Why it's incredible: The giant meadow formed from a single seedling.

The crystal-clear waters of Shark Bay are home to the world's largest plant: a seagrass meadow spanning 77 square miles (200 square kilometers) and stretching 112 miles (180 kilometers) from end to end. The shoots that make up the massive meadow all originate from one stem, which researchers estimate is at least 4,500 years old.

Researchers analyzed the genetic makeup of the seagrass at Shark Bay for the first time in 2022. They discovered that almost all the Poseidon's ribbon weed (Posidonia australis) which grows in a network of meadows inside the bay was genetically identical, indicating that the weeds belonged to one plant. Unlike other types of seagrass, which reproduce sexually, this meadow appeared to be continually cloning itself through an underground stem called a rhizome.

Related: 2 plants randomly mated up to 1 million years ago to give rise to one of the world's most popular drinks

On the sandy seafloor, the self-cloning seagrass meadow forms clumps of grass that look like separate organisms, but underground, the shoots are connected to form "the largest clone in any environment on Earth," the researchers wrote in the study.

This means the Shark Bay seagrass dwarfs the previous record-holder: The second-largest clone on record is a 9-mile-long (15 km) meadow of Posidonia oceanica seagrass in the western Mediterranean Sea.

The meadow at Shark Bay is expanding through a process known as "horizontal rhizome extension," in which the plant grows stems that extend horizontally beneath the seafloor. These stems then grow vertical stems that develop shoots and leaves, which break through the sand to form seagrass. Based on the size and age of the meadow, researchers estimate it is expanding by around 6 to 14 inches (15 to 36 centimeters) per year which is fairly quick compared with other self-cloning seagrass meadows, according to the study.

The meadow has remained relatively undisturbed for millennia, which has enabled it to reach colossal proportions. The clone could continue its creeping expansion for as long as it is left untouched, making it practically immortal, Elizabeth Sinclair, an evolutionary biologist at the University of Western Australia, previously told Live Science.

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Shark Bay: Home to Earth's largest plant an immortal, self-cloning seagrass meadow stretching 112 miles - Livescience.com

Levine is an election integrity and management consultant who works to ensure that eligible voters can vote, free and fair elections are perceived as legitimate, and election processes are properly administered and secured.

Imagine its Election Day. Youre getting ready to go vote when you receive a call from a public official telling you to vote at an early voting location rather than your Election Day polling site. So, you go there only to discover its closed. Turns out that the call wasnt from the public official but from a replica created by voice cloning technology.

That might sound like something out of a sci-fi movie, but many New Hampshire voters experienced something like it two days before the 2024 presidential primary. They received robocalls featuring a deepfake simulating the voice of President Joe Biden that discouraged them from participating in the primary.

To be sure, theres no indication that the fake Biden robocalls had a discernible impact on the New Hampshire primary, but the incident is a stark reminder of the growing threat posed by tactics like this, which are increasingly being used by malign actors to target elections not only in the U.S. but in Slovakia, Argentina and elsewhere.

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As artificial intelligence tools become more accessible and affordable, deepfake attacks (of which voice cloning is only one example) are becoming more frequent. How can voters protect themselves from similar efforts to ensure that they make informed decisions for the November general election? Here are a few tips:

1.Avoid answering calls from unknown numbers: Picking up a call from an unknown number increases the likelihood of falling for a scam. Additionally, if you answer a call from an unknown number and speak, a scammer can record your voice and use it to create cloned scam calls to trick your family members and friends.

2.Verify the callers identity: If you do answer a call that raises suspicion, take steps to verify the callers identity. Several New Hampshire votersdid this after receiving the Biden robocall and were able to confirm that the voice was fake. Try to contact the person (or their campaign) through an alternative channel to confirm that the call was actually from the person/organization it purported to be from.

3.Report potential voice cloning: If you may have received an AI voice scam call, contact the appropriate authorities so they can use their expertise to investigate further. This can help address your scam, as well as others, and deter similar future behavior. After New Hampshire voters alerted law enforcement and their attorney general about the robocall that used AI to impersonate Biden, the alleged culprit was identified and charged with 13 counts of voter suppression, a felony, and 13 counts of impersonating a candidate, a misdemeanor. He also faces a proposed $6 million fine from the Federal Communications Commission.

4.Educate yourself: Knowledge is your best defense against emerging threats. Take the time to educate yourself and those around you about the dangers of voice cloning. Be skeptical of unsolicited calls, especially if they involve urgent requests that offer suspicious information or try to get you to engage in behavior that sounds off (like sending gift cards to supposed relatives of friends).

5.Rely on trusted sources: Our information ecosystem is awash in lies and inaccurate information, but at least in the elections space we know whom to seek out for accurate information about the administration of elections: state and local election officials (and those who support their efforts).

6.Make a plan to vote in advance of Election Day: Devising a vote plan allows you to confirm when, where and how you can vote. It also enables you to consider alternatives in case your preferred plan for voting does not work out because of something unforeseen like an illness. Finally, planning makes it less likely that youll be tricked by something like a voice cloning attack, even if it appears real.

Voice cloning attacks are part of the new frontier in malign efforts to meddle in U.S. elections. By staying informed, establishing safeguards, and remaining skeptical of unexpected communications, voters can increase their chances of thwarting these threats before they cause real damage.

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Overcoming AI voice cloning attacks on election integrity - The Fulcrum

Today is Friday, July 5, the 187th day of 2024. There are 179 days left in the year.

Todays Highlight in History:

On July 5, 1996, Dolly the sheep, the first mammal cloned from an adult somatic cell by scientists at the Roslin Institute at the University of Edinburgh, was born.

Also on this date:

In 1687, Isaac Newton first published his Principia Mathematica, a three-volume work setting out his mathematical principles of natural philosophy.

In 1811, Venezuela became the first South American country to declare independence from Spain.

In 1852, Frederick Douglass delivered his speech What to the Slave is the Fourth of July? at Corinthian Hall in Rochester, New York.

In 1865, the Secret Service Division of the U.S. Treasury Department was founded in Washington, D.C., with the mission of suppressing counterfeit currency.

In 1935, President Franklin D. Roosevelt signed the National Labor Relations Act.

In 1937, Hormel introduced a canned meat product called Spam; more than 9 billion cans have been sold since.

In 1940, during World War II, Britain and the Vichy government in France broke off diplomatic relations.

In 1943, the Battle of Kursk began during World War II; in the weeks that followed, the Soviets were able to repeatedly repel the Germans, who eventually withdrew in defeat.

In 1946, the modern bikini, designed by Frenchman Louis Reard, was first modeled in Paris.

In 1947, Larry Doby made his debut with the Cleveland Indians, becoming the first Black player in the American League three months after Jackie Robinson broke the color barrier in the National League.

In 1954, Elvis Presley recorded his first single, Thats All Right, at Sun Studio in Memphis, Tennessee.

In 1971, President Richard Nixon certified the 26th Amendment to the U.S. Constitution, which lowered the minimum voting age from 21 to 18.

In 1975, Arthur Ashe became the first Black man to win a Wimbledon singles title, defeating Jimmy Connors.

In 1977, Pakistans army, led by General Mohammad Zia ul-Haq, seized power from President Zulfikar Ali Bhutto.

In 1980, Bjorn Borg became the first male player to win five consecutive Wimbledon singles titles.

In 1994, Amazon was founded by Jeff Bezos as an online marketplace for books.

In 2011, a jury in Orlando, Florida, found Casey Anthony, 25, not guilty of murder, manslaughter and child abuse in the 2008 disappearance and death of her 2-year-old daughter, Caylee.

In 2013, Pope Francis cleared two of the 20th Centurys most influential popes to become saints in the Roman Catholic church, approving a miracle needed to canonize Pope John Paul II and waiving Vatican rules to honor Pope John XXIII.

Todays Birthdays: Julie Nixon Eisenhower is 76. Rock star Huey Lewis is 74. Baseball Hall of Fame pitcher Rich Goose Gossage is 73. NFL Hall of Fame receiver James Lofton is 68. Cartoonist Bill Watterson (Calvin and Hobbes) is 66. Singer-songwriter Marc Cohn is 65. Actor Edie Falco is 61. Actor Jillian Armenante is 60. Actor Kathryn Erbe is 59. Actor Michael Stuhlbarg is 56. Rapper RZA (RIH-zuh) is 55. Author Gary Shteyngart is 52. R&B singer Joe is 51. Rapper Royce da 59 is 47. International Tennis Hall of Famer Amelie Mauresmo is 45. Actor Ryan Hansen is 43. Country musician Dave Haywood (Lady A) is 42. Actor Danay Garcia is 40. Soccer player Megan Rapinoe is 39. Actor Jason Dolley is 33. Los Angeles Dodgers pitcher and designated hitter Shohei Ohtani is 30.

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Today in History: Dolly the sheep marks cloning breakthrough - Chicago Tribune

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Cloned food is entering Canadian markets without mandatory labelling

Health Canadas recent round of consultations, conducted with minimal public awareness, suggests that these products may soon be available without consumers knowledge, as there will be no mandatory labelling. The absence of such information on Health Canadas website only adds to the opacity surrounding cloned products.

The consultation, which concluded on May 25th, focused on updating the Policy on Foods Derived from Cloned Animals by Somatic Cell Nuclear Transfer and Their Progeny. This update proposes that cloned products be exempt from pre-market evaluation under Part B, Title 28 of the Food and Drug Regulations. This approach differs from other nations like the United States, Japan, and New Zealand.

But what precisely is animal cloning? The process aims to create a genetic replica of an animal by replacing the nucleus of an unfertilized egg with that of a somatic cell from the donor animal, forming an embryo. This embryo is then implanted into a surrogate mothers uterus, where it develops to term.

Artificial insemination, a well-established industry practice, involves collecting sperm from a male and artificially introducing it into a females reproductive system to facilitate fertilization, preserving genetic variability. Cloning, however, produces genetically identical animals, eliminating this variability.

From a food safety perspective, cloned products do not pose a threat to human health. However, the social and moral acceptability of cloning remains in question. It is doubtful that consumers will unconditionally accept this technology, especially in the absence of labelling. For traditional producers, integrating cloned products into the market could also taint consumer perceptions across entire categories, particularly meat and dairy.

This situation mirrors the backlash against genetically modified salmon, which faced immediate retail rejection despite being deemed safe. Irrespective of the safety profile, it is crucial to explain the technology and ensure consumers comprehend the rationale and necessity for such practices, both for their benefit and that of the industry.

For the industry, the imperative to amend regulations is less evident. Cloning is an expensive process, and the argument that reduced production costs will translate into lower retail prices for consumers is tenuous at best.

Without mandatory labelling, offering consumers a truly informed choice becomes problematic. We have witnessed similar issues with genetic engineering and GMOs. Health Canada appears poised to embrace technological advancements impacting our agri-food sector without adequately considering consumer rights and preferences.

Quite shameful.

Dr. Sylvain Charlebois is senior director of the agri-food analytics lab and a professor in food distribution and policy at Dalhousie University.

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The opinions expressed by our columnists and contributors are theirs alone and do not inherently or expressly reflect the views of our publication.

Troy Media Troy Media is an editorial content provider to media outlets and its own hosted community news outlets across Canada.

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Cloned meat on the menu? - Troy Media