#clone PCB

[pcbreverseengineering]

Understanding the Process and Benefits of Clone PCB

A clone PCB refers to a printed circuit board that replicates the design of an existing board. This concept can be crucial for a variety of applications, including reverse engineering, prototype development, and even product duplication. In this article, we'll delve into what it means to clone a PCB, the process involved, and the benefits it can bring.

What is a Clone PCB?

In essence, a Clone PCB is a duplicate of an authentic printed circuit board (PCB). This copying process can be used for several reasons, such as creating a backup of a critical component, manufacturing a similar product, or conducting repairs on outdated electronics . Cloning a PCB involves duplicating its design and functionality while maintaining the same electrical characteristics and performance as the original board.

The Process of Cloning a PCB

Cloning a PCB involves several key steps to ensure that the final product matches the original board's specifications. This is a condensed explanation of the procedure:

Design Analysis : The first step in creating a clone PCB is to thoroughly analyze the design of the original board. Examining the design, component positioning, and electrical connection routing are all part of this. Advanced tools like scanning software and schematic capture programs can be used to capture and replicate these details accurately.

Reverse Engineering : After design analysis, the next phase is reverse engineering. This involves creating a detailed schematic and layout based on the analysis of the original PCB. Reverse engineering tools can help convert physical PCB designs into digital formats, making it easier to replicate the board.

Fabrication and Assembly : With the digital design files ready, the clone PCB can be fabricated. This involves printing the PCB design onto a substrate, etching away excess material, and then placing and soldering components onto the board. Quality control is crucial during this phase to ensure that the cloned board meets the original specifications.

Testing and Validation : Once the Clone PCB is assembled, it undergoes rigorous testing to verify that it functions as intended. This includes checking electrical performance, signal integrity, and overall reliability. The goal is to ensure that the cloned PCB performs identically to the original board in every aspect.

Benefits of Cloning a PCB

Cost-Effective Solutions : One of the primary benefits of a clone PCB is cost savings. Cloning can be a more affordable option compared to designing a new PCB from scratch, especially when replicating an existing, proven design.

Prototyping and Testing: For engineers and developers, cloning a PCB allows for rapid prototyping and testing. This can be especially useful for developing new products or testing modifications without the need for extensive redesign efforts.

Replacement for Obsolete Components: As technology evolves, certain PCBs and their components may become obsolete. Cloning a PCB provides a viable solution for replacing or repairing outdated electronics, ensuring continued functionality of critical systems.

Educational Purposes: Cloning PCBs can also serve educational purposes. It offers students and enthusiasts the opportunity to learn about PCB design and assembly by working with real-world examples.

Product Duplication: For manufacturers, cloning a PCB allows for the duplication of successful products. This can be beneficial for scaling production or creating variations of a popular design.

The process of creating a clone PCB involves a detailed and meticulous approach to ensure accuracy and functionality. By understanding and utilizing the cloning process, businesses and individuals can enjoy the benefits of cost-effective solutions, enhanced prototyping, and the ability to maintain and repair obsolete electronics. Whether for educational purposes, product duplication, or practical applications, cloning a PCB provides valuable opportunities in the world of electronics.

[perlina-hitechcircuits]

Hitech Circuits Provides Reliable PCB Clone Service

Hitech Circuits Electronic boasts nearly two decades of experience in the PCB industry. In addition to offering PCB design and assembly services, we specialize in providing reliable PCB Clone services. It’s crucial to note that while Clone can be legal under certain circumstances, it might infringe on intellectual property rights or breach contractual agreements in some cases. Therefore, we emphasize the importance of thoroughly assessing and understanding the legal implications associated with this process.

At Hitech Circuits Electronic, we adhere to ethical practices and ensure that our Clone projects comply with all legal boundaries. Our goal is to provide fully functional replacements that can repair, replicate, or enhance the capacity of your outdated electronics while respecting intellectual property rights. Contact our team today to start your custom PCB Clone project and benefit from our years of expertise in the field. If you need the service of PCB clone, please feel free to contact me: sales3@hitechpcb.com

[icunlockmcucrack]

PCB Clone Services: Precision in Reproduction

What is PCB Cloning?

PCB Clone is the process of creating an exact replica of an existing printed circuit board (PCB). This service is essential when you need to reproduce a board for which the original design files are unavailable. PCB cloning involves reverse engineering the board to generate the necessary data for manufacturing a duplicate.

The Role of PCB Clone Services

PCB Cloning Service are vital in scenarios where you need an identical copy of an electronic circuit board. Whether it’s for replacing obsolete hardware, producing additional units, or creating backups, these services ensure precision in reproduction.

How PCB Cloning Works

1. Initial Analysis

The cloning process begins with a detailed analysis of the original PCB. This includes examining the board’s layout, components, and circuitry. Engineers carefully inspect every detail to ensure accurate replication.

2. Reverse Engineering

After the initial analysis, the reverse engineering phase begins. Engineers trace the circuitry, identify components, and create a schematic of the board. This step is crucial in understanding how the PCB functions.

3. Schematic Capture and Layout Design

Once the reverse engineering is complete, a schematic diagram is created. This diagram serves as a blueprint for the new board. Engineers then use specialized software to design a PCB layout that matches the original.

4. Prototyping and Testing

Before full-scale production, a prototype of the cloned PCB is manufactured. This prototype undergoes rigorous testing to ensure it functions exactly like the original board. Any necessary adjustments are made during this phase.

5. Mass Production

Once the prototype passes all tests, the PCB is ready for mass production. The final product is an exact clone of the original board, ready for use in any application.

Benefits of PCB Cloning Services

• Obsolete Hardware Reproduction: Clone services are ideal for replicating outdated circuit boards, ensuring continued operation of legacy systems.

• Cost-Effective: Cloning is often more cost-effective than redesigning a PCB from scratch.

• Fast Turnaround: PCB cloning services can quickly produce duplicates, reducing downtime for critical systems.

Challenges in PCB Cloning

PCB cloning, while beneficial, is not without its challenges. Complex multilayer boards, proprietary components, and encrypted firmware can complicate the cloning process. Skilled engineers are essential to navigate these obstacles and deliver an accurate clone.

Choosing the Right PCB Clone Service

When selecting a Clone PCB Service, consider factors such as experience, technology, and the ability to handle complex projects. A reliable service provider will ensure that your cloned PCBs meet the highest standards of quality and performance.

Conclusion

PCB cloning services play a crucial role in the electronics industry. Whether for preserving legacy systems or producing additional units, these services provide a reliable solution for duplicating PCBs with precision. With the right expertise, PCB cloning ensures that your electronic systems continue to function smoothly, regardless of the challenges posed by outdated or unavailable designs.

[fastpcbcopy]

Clone Pcb Board in France

Discover fastpcbcopy, your go-to destination for cloning PCB boards in France Specializing in precise replication, we guarantee top-notch quality and confidentiality. Break barriers and innovate with us today! Contact fastpcbcopy for seamless solutions customized to your requirements.

[circuit-engineer]

PCB Cloning Services: What You Need to Know

Introduction

In the fast-paced world of electronics, staying ahead of the competition often requires innovation and efficiency. PCB cloning services play a pivotal role in achieving this goal. If you're in need of PCB reverse engineering, cloning, or copying services, Circuit Engineering Company Limited is your go-to destination. Explore the world of PCB cloning and discover the exceptional services offered by Circuit Engineering.

What is PCB Cloning?

PCB cloning, also known as PCB reverse engineering, is the process of recreating an existing printed circuit board (PCB) design without access to the original design files. This service is invaluable for various purposes, including product improvement, quality control, and overcoming obsolescence issues. Circuit Engineering specializes in providing top-notch PCB cloning services.

Understanding PCB Cloning Services at Circuit Engineering

Expertise and Experience

Circuit Engineering boasts a team of skilled professionals with years of experience in PCB cloning. They have successfully worked on a wide range of projects, from simple single-layer boards to complex multi-layer designs. This expertise ensures that you'll receive a high-quality and accurate cloned PCB.

State-of-the-Art Technology

Circuit Engineering uses cutting-edge technology and equipment to ensure precision and reliability in the PCB cloning process. Advanced tools and software are employed to analyze and recreate the PCB design, ensuring that the cloned board functions as expected.

Quality Assurance

Quality is paramount in the world of PCB cloning. Circuit Engineering is committed to delivering PCB clones that meet or exceed industry standards. Rigorous testing and quality assurance processes are in place to ensure the reliability and durability of the cloned PCBs.

Cost-Effective Solutions By choosing PCB cloning services from Circuit Engineering, you can save both time and money. Instead of redesigning an entire PCB from scratch, cloning allows you to replicate an existing design accurately, reducing development time and costs.

Versatility

Circuit Engineering's PCB cloning services are highly versatile. Whether you need to clone a single PCB or an entire product line, their team can handle the task efficiently. Their services are suitable for industries such as consumer electronics, aerospace, automotive, and more.

Why Choose Circuit Engineering?

Customer-Centric Approach: Circuit Engineering focuses on understanding your unique requirements and tailoring their services to meet your needs.

Timely Delivery: They recognize the importance of deadlines and work diligently to ensure on-time delivery of cloned PCBs.

Confidentiality: Your intellectual property is safe with Circuit Engineering. They maintain strict confidentiality throughout the cloning process.

Affordable Pricing: Circuit Engineering offers competitive pricing, making their PCB cloning services accessible to a wide range of customers.

Conclusion

If you're in need of PCB cloning, reverse engineering, or copying services, Circuit Engineering Company Limited is your trusted partner. Their expertise, state-of-the-art technology, commitment to quality, and cost-effective solutions make them the best choice for your PCB cloning needs. Discover the world of PCB cloning with Circuit Engineering, and unlock the potential for innovation and efficiency in your electronics projects.

For more information and to explore their services, visit https://www. .com today. Your journey towards top-quality PCB cloning starts here.

Contact Us Circuit Engineering Company Limited

Rm.1902, Easey Comm. Bldg., 253-261 Hennessy Road, Wanchai, Hong Kong. Tel: 86-755-21019972

Fax: 86-755-21019976

Skype: circuitengineer

Whatsapp: 86-13430727446

Contact us via email at: infocircuitengineer.com

[fuzzkaizer]

PHEADHONES - Noise Swash clone

"... of the 4ms pedal using a custom-etched PCB also by acidrainpedalworks"

cred: pheadhones.tumblr.com

[kfcdoubledown]

Sadly I must report that when the fucking thing actually works and has a screen that isn't smashed, the Retroid Pocket 2S is a really top shelf handheld. Sticks and triggers are good (and hall effect), d-pad is a clone of the PS Vita's so it feels like sex, membrane buttons on ABXY are great, and all emulators are set up for you out of the box.

Convoluted PCB on the inside, though, two cables run along the back of the screen and the battery rests on top of them, and the screen is essentially fused to the front shell so Retroid has to sell or replace screens as a full front shell swap. The right side PCB half (controls d-pad, L buttons and left stick) bumps into the shell's retention clips if you try to take it out, it's a tight fit. Mysteriously, fixing the screen actually did fix the HDMI output.

[perfecttenth]

Perfect Origin submissions and beta designs

Or at least what's known about them.

Starting at zero, The Man's concept art has been uploaded to Pixiv by his creator, Seiji Tanaka, here and here. He was originally submitted as "Zeusman", based on the Greek deity of the same name. Even back then, he was supposed to be the true identity of Strong The Budo, except that in this rendition, he is a choujin cloned from the cells of Neptune King.

While Goldman was likely created by Yudetamago themselves, his Akuma Shogun identity is a refined version of "Vikingman", who was submitted by Kobayashi of Tokyo. Early color illustrations of Shogun tend to depict him with pure red armor, which may have been a leftover from his submission.

Silverman's redesign process for his appearance in the 2011 run was extensively documented and released in the "JC55巻 56巻 発売記念品 2016 夏" booklet. His first rough design is completely different from the finished product, mirroring Shogun even moreso than his final design.

Mirageman is one of the two Kenichi Ishii-submitted Origins, the other being Justiceman. Both him and Thingman, designed by Ken Hiroshima (who also worked on Painman together with Kentaro Sakamoto), were first shown as choujin submissions in Vol. 25 of Nisei Tag, named "Mirage" and "Doppelman" respectively. Thingman is nigh identical to his submission, but Mirageman used to somewhat resemble a circuit board, with PCB traces and pads covering his body.

Abyssman is credited to Masashi Omori and Takamasa Minato, presumably being a combination of multiple submissions, as the Fan Book commentary implies.

Ganman is famous for not only being a combination of two submissions, but also having his appearance modified after his initial reveal. His moose features were taken from a currently unknown submission that were applied to WANI's "The Cyclone".

Yudetamago later clarified in the Vol. 47 Q&A that his design was upgraded to make him look more dignified, as he's supposed to be on the same level as Akuma Shogun. I've uploaded a catbox album with all the panels I could find of Ganman's original appearance here, but the original WEB version of the chapter can't be found in its entirety anymore.

Crowman/Karasuman was submitted by Takayuki Wakisaka. His name and design have stayed exactly the same, except he's way thinner here than in the manga.

The same Takayuki Wakisaka also appears to have submitted Psychoman's Grim Reaper identity. His new white priest-like design was probably refined by Yudetamago, since an Instagram post by Takashi Shimada shows what looks like concept art, as well as an alternate design.

As of right now, this is all that is known about the design processes of the Perfect Origin. Feel free to add or correct anything if you happen to have more information.

[technician-the]

Two of the three PCB's that make up the piece of rack gear I call my DI box.

on the left, is a clone of the EQ section from a 70s Sunn Coliseum Bass amp, Half assembled.

It is a 4 knob eq, with controls idiosyncratically (but accurately) labeled high, mid, hi bass, and low bass. the high and mid controls each have a 2 position frequency switch. The result is a very versatile, but still easy to tune tone control.

On the right is my standard line output pcb, fully assembled, except for the gain setting resistor.

this is a transformer coupled design, which uses a small power amp, to ensure it cant be overloaded. I didn't know it when I designed this, but this is almost identical to the design used by urei in the late 1970s, notably in the 1178, and la4a.

It has better isolation and a lower noise floor then a modern integrated design, and is actually cheaper then the IC solutions sold by That crop.

I used the same pcb with a different transformer as a reverb driver in my rack mount spring reverb.

If you have ever listened to my music you have heard both these circuits on synth, guitar, bass, and even my drum machines. I've also sold several identical units to local studios.

also, the spring reverb is my go to reverb now. So its on most tracks as well.

[pheadhones]

my collection of pedals with acid-etched artwork done by my friend Russell (ig: @ acidrainpedalworks)

top: Digital Scream & ASD, my original designs

mid: Judas & Super Soda Jerk, an original circuit and Devi Ever clone/tribute by acidrainpedalworks

bottom: Noise Swash, clone of the 4ms pedal using a custom-etched PCB also by acidrainpedalworks

[dandelionsresilience]

Good News - April 22-28

Like these weekly compilations? Support me on Ko-fi! Also, if you tip me on here or Ko-fi, at the end of the month I'll send you a link to all of the articles I found but didn't use each week - almost double the content! (I'm new to taking tips on here; if it doesn't show me your username or if you have DM's turned off, please send me a screenshot of your payment)

1. Millions Of Maggots Donated To Charity In Honour Of The Maggot King

“[Blizzard Entertainment] donated millions of maggots to Tiggywinkles UK, a leading wildlife hospital, […] in honor of Duriel, the Maggot King, a notorious character in the Diablo series. […] This significant contribution is expected to feed the patients at Tiggywinkles for two years.”

2. New circuit boards can be repeatedly recycled

“A team led by researchers at the University of Washington developed a new PCB that performs on par with traditional materials and can be recycled repeatedly with negligible material loss. Researchers used a solvent that transforms a type of vitrimer—a cutting-edge class of sustainable polymers—to a jelly-like substance without damaging it, allowing the solid components to be plucked out for reuse or recycling.”

3. How a Cloned Ferret Inspired a DNA Bank for Endangered Species

“This black-footed ferret (Mustela nigripes) [is] the first endangered species in the U.S. to ever be successfully cloned […] from [a] cryogenically preserved cell line, obtained from a ferret named Willa, who lived in Wyoming in the 1980s. [… T]he FWS [is now] on a major project to cryogenically store tissue from every endangered species in the U.S., [… as] “an insurance policy against future loss of biodiversity in the wild.””

4. Maine Signs Trans And Abortion Sanctuary Bill Into Law, Despite Violent Threats

“[The new law] states that criminal and civil actions against providers and patients are not enforceable if the provision or access to that care occurred within Maine’s borders, asserting jurisdiction over those matters. It bars cooperation with out-of-state subpoenas and arrest warrants for gender-affirming care and abortion that happen within the state.”

5. $70M for Fish Passage Projects Address Climate Resilience, Strengthen Local Economies

“The Department of the Interior today announced more than $70 million […] that will improve fish passage around […] barriers fragmenting the nation’s rivers and streams [… as an] “investment to restore our nation’s rivers and streams, safeguard endangered fish species, protect Indigenous subsistence practices and provide communities with increased opportunities for outdoor recreation and economic growth,” said Secretary Deb Haaland.”

6. A Golden Age of Renewables Is Beginning, and California Is Leading the Way

“California—the fifth-largest economy in the world—has experienced a record-breaking string of days in which the combined generation of wind, geothermal, hydroelectric and solar electricity has exceeded demand on the main electricity grid for anywhere from 15 minutes to 9.25 hours per day.”

7. Collaborative conservation efforts keep lake sturgeon off the endangered species list

“The Service’s 12-month finding [that these sturgeon are not endangered] shows ongoing management efforts, such as fish stocking, have contributed to the conservation and resiliency of the species. “Today’s announcement shows the power of collaborative conservation and the impact it can have for species like the lake sturgeon,” said Midwest Regional Director Will Meeks.”

8. French municipalities bordering Luxembourg to offer free transport to commuters

“Luxembourg is the only country in the world to offer entirely free public transport. […S]ix French communities located near the border and many of whose residents work in Luxembourg have decided to band together and also offer a free shuttle for daily workers. […] Last year, governments in the two countries said they would work to increase train services to run every 10 minutes between Metz and Luxembourg City by 2030. There’s also a plan to extend Luxembourg City’s tram service to reach all the way to the border – a trip that would take only 30 minutes when ready.”

9. Solar is about to get a lot more affordable for low-income households

“The EPA granted 49 state and territory awards totaling $5.5 billion, six tribal awards worth more than $500 million, and five multistate awards amounting to $1 billion. Together, the awards cover all 50 states, Puerto Rico, and the District of Columbia. All of the funding is dedicated to low-income and disadvantaged households.”

10. Amsterdam will cover cost of changing one's gender on documents

“[T]he city’s residents will be able to change the gender on their birth certificate and Basic Registration of Persons (BRP) free of charge. [… T]he local government will also reimburse (part of) the costs of a new passport, identity card or driver's license.”

April 15-21 news here | (all credit for images and written material can be found at the source linked; I don’t claim credit for anything but curating.)

[pcbreverseengineering]

Note the Various Benefits of Clone PCB

A clone PCB is a replicated version of an existing printed circuit board (PCB), designed to mimic the layout, components, and functionality of the original PCB. It serves purposes such as backup, legacy support, cost reduction, customization, or reverse engineering. However, creating clone PCBs must be done while considering legal and ethical considerations regarding intellectual property rights.

Sometimes, clone PCBs are created through reverse engineering processes to replicate the functionality of an existing PCB without access to its original design files or documentation. PCB (Printed Circuit Board) reverse engineering is the process of deconstructing and analyzing the design and functionality of a printed circuit board to understand its schematics, layout, and operation.

There are multifaceted benefits of clone PCBs:

Continuity and redundancy - Clone PCBs provide a safety net for businesses by ensuring continuity in production processes. In case the original PCB design becomes unavailable due to damage, loss, or obsolescence, having clone PCBs allows manufacturing to continue without disruptions. This redundancy is crucial for industries where downtime can result in substantial financial losses.

Legacy support and maintenance - Many industries rely on equipment or devices with legacy PCB designs that may no longer be supported by the original manufacturer. Clone PCBs enable the continued operation, maintenance, and repair of such equipment, extending its lifespan and maximizing return on investment. This is particularly important in sectors like aerospace, defense, automotive, and industrial manufacturing, where equipment longevity is paramount.

Cost optimization - Clone PCBs offer opportunities for cost optimization in several ways. By replicating the original PCB design using more readily available or cost-effective components, manufacturers can reduce production costs without sacrificing quality or performance. Additionally, economies of scale can be leveraged when producing clone PCBs in bulk, further driving down per-unit costs.

Flexibility and customization - Clone PCBs can be tailored to meet specific requirements or to incorporate improvements over the original design. Manufacturers can modify aspects such as component selection, layout, or functionality to optimize performance, enhance reliability, or adapt to evolving technological standards. This flexibility enables businesses to stay competitive and responsive to changing market demands.

Reverse engineering and innovation - Clone PCBs support innovation by facilitating reverse engineering efforts. Analyzing and replicating existing PCB designs allows engineers to gain insights into complex electronic systems, identify best practices, and develop new products or technologies based on proven designs. This process fosters creativity, knowledge transfer, and continuous improvement within the industry.

Intellectual property protection - While not directly a benefit, clone PCBs can also help protect intellectual property (IP) by offering a legal and authorized means for manufacturers to reproduce their own designs. This ensures that valuable innovations are safeguarded against unauthorized replication or counterfeiting, preserving the competitive advantage of original equipment manufacturers.

[metsn]

WAR HEAD - a versatile fuzz with massive boost

WAR HEAD is a versatile fuzz based on a clone of an obscure BIG MUFF clone, that is also known as the very popular FUZZ WAR. Additionally these METSÄÄN machines also contain a massive booster circuit (based on the EHX LBP1) to give you the full blast of signal shredding in one pedal. The top left pot is the input GAIN and the one right of that is the output VOLUME pot. Below those in the middle is the TONE control, ranging from a deep low end rumble to tissue slicing high frequencies. It is a TRUE BYPASS effect with a green indicator LED, build into a die-cast aluminum enclosure painted with an unique ‘burned out industry’ camo pattern and a piece of PCB around the knobs. Power-socket: 9V DC , 2,1mm DC jack boss-style (inside -/+ outside) PSU is NOT included. Handmade by GRM for METSÄÄN. Shipping from finland. BUY HERE. (link to new webshop) Here's a little video clip of the WAR HEAD in action: https://www.instagram.com/reel/DBWSksjt3uZ

[aurianneor]

Cancer, give people a fighting chance

The cells of the human body have a life span of 2 weeks to 15 years. To ensure the continuity of the human body, they duplicate and then die. This is written into the DNA. Hormones trigger DNA functions. There are a hundred thousand billion cells in the human body. Every day, 50 billion cells must clone themselves with their DNA and die.

Sometimes errors occur during DNA duplication. Most of the time, the new cell will have a blueprint foreign to the human body. The immune system will destroy it. The DNA may have an error, but the hormones don’t activate it. There are no consequences. The DNA may be incorrectly transcribed, the cell will malfunction, but it is recognized as part of the human body and reproduces with its defects. These errors occur 45 million times a day. That’s old age.

Once in a while, a mistake is made and the cell forgets to die. It becomes immortal. It clones itself, and instead of having one new cell and one that dies, there are two. Then four, then sixteen, and so on. This is cancer. Cells malfunction and increase in number instead of remaining constant.

Cancer is dangerous because these cells upset the balance of the human body: too many hormones, too many bones, too much stomach, and so on. When they take up too much space, we call it a tumor. The distribution of cancers in the population is linear, typical of a statistical distribution, i.e. cancer is a function of chance. Game theory says that if you roll a dice enough times, you’ll end up with a given number. If there are 45 million mistakes a day, the chances of these mistakes becoming cancer increase with the number of repetitions, and therefore with age. But they can also affect a child or a baby; it’s rarer, but it does happen. Once you reach a certain age, it’s almost certain, and it can appear in any cell of the body. The first factor that could lead to cancer one day is statistical.

Deviations from this statistical curve range from 5 to 10%. Lifestyle has little impact. Drinking alcohol, sun exposure and smoking increase the chances of getting cancer, but not by much.

Most Cancer Is Beyond Your Control, Breakthrough Study Finds – Time: https://time.com/3650194/most-cancer-is-beyond-your-control-breakthrough-study-finds/

What causes a cell to become cancerous is first and foremost DNA transmission errors, 99% of which are due to radiation. The sun bombards us with radioactivity every day. Soil, water and therefore food contain radioactive elements.

Cells are specialized. If the erroneous transmission occurs, but the cell does not use this function, this part of the DNA will not cause cancer.

Hormones are the subsequent cause of a cell becoming cancerous. When hormones don’t work properly, they ask the cell to use the DNA function that doesn’t correspond to it. There are external agents that behave like hormones. These are known as endocrine disruptors. Everything derived from fossil fuels (plastics, oil, uranium, etc.) comes from living matter containing hormones. These hormones have the ability to behave like human hormones. One day, one of these hormones touches a cell and activates its DNA. This increases the risk of cancer by a factor of 5 to 10%.

Evaluating intrinsic and non-intrinsic cancer risk factors – nature communications: https://www.nature.com/articles/s41467-018-05467-z

Since 1990, the number of cancers diagnosed worldwide has risen by 75%. A billion Chinese and a billion Indians have beaten infectious diseases, so they live longer and die of cancer more often. The chances of developing cancer under the age of 50 have fallen.

Change in three measures of cancer mortality, World, 1990 to 2019 – Our world in data: https://ourworldindata.org/grapher/cancer-deaths-rate-and-age-standardized-rate-index

There are some endocrine disruptors that have not been used since the 90s: PCBs and DDTs. They polluted the whole world. Most of the endocrine disruptors we receive come from the air, from atmospheric pollution. For example, someone living in Beijing is just as likely to get cancer as someone who smokes 25 cigarettes a day.

If you live in a big city you already smoke every day– World economic forum: https://www.weforum.org/agenda/2018/05/if-you-live-in-a-big-city-you-already-smoke-every-day/

We can remove the endocrine disruptors used in factories for the profits of a few: pesticides, petroleum, plastic, the use of fire etc. This would reduce the risk of cancer somewhat, as it would reduce DNA activation in poorly transcribed cells.

What really makes a difference is that medicine is able to make people with cancer live longer. For breast cancer, for example, there was a 45% chance of survival over 10 years in 1971. Today, it has a 78% chance of being alive in 10 years.

It’s important that there should be real public research into cancer. Survival is a direct result of medicine. We can live much better with cancer.

Dietary supplements, antioxidants and spa treatments have no effect on cancer, but they do capture a huge amount of money that is not invested in science.

Some scientists suggest renaming cancers to avoid using names of areas of the body in which it has been detected, whereas cancer spreads throughout the body. Instead, they recommend using the nature of the cancer and the way it spreads.

We’re naming cancers all wrong, oncology leader says – Stat News: https://www.statnews.com/2024/01/31/oncology-name-cancer-by-genetics-not-organs-expert-says/

Updating the Definition of Cancer – NIH: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10618731/

You can’t fight cancer. You can’t blame the person for getting cancer. On the other hand, you have to accept medical care. Infectious diseases have been beaten with antibiotics, not by making victims feel guilty.

Let King Charles’s illness finally change how we speak about cancer: it’s not about ‘winning’ or ‘losing’ a ‘war’ – The Guardian: https://www.theguardian.com/commentisfree/2024/feb/16/king-charles-speak-cancer-winning-losing-war-bowel-language

CAR-T cell therapy is a new invention. Emilie Whitehead was the first person to be treated with this therapy. At the age of 7 in 2012, when she was dying, her doctors used the new CRISPR CAS-9 technique to modify her immune cells to fight her own cancer. Ten years on, Emilie is still alive and enjoying a normal life.

he Emperor of All Maladies: A Biography of Cancer – Siddhartha Mukherjee: https://www.goodreads.com/book/show/7170627-the-emperor-of-all-maladies

The Song of the Cell: An Exploration of Medicine and the New Human – Siddhartha Mukherjee: https://www.goodreads.com/book/show/60321392-the-song-of-the-cell

------------------------------------------------------------------------

Cancer, aider le hasard: https://www.aurianneor.org/cancer-aider-le-hasard/

Tous égaux face au cancer du sein: https://www.aurianneor.org/tous-egaux-face-au-cancer-du-sein/

Découvrez la dernière anecdote via @Sciencetips_fr : “Il a du flair !” cancer ?? ??: https://www.aurianneor.org/decouvrez-la-derniere-anecdote-via-sciencetipsfr/

Change the words “HIV” and “AIDS” by “Mental illness” and see how you feel about it: https://www.aurianneor.org/change-the-words-hiv-and-aids-by-mental/

In yogurt we trust: https://www.aurianneor.org/crispr-cas9-a-single-shot-to-cure-a-disease-that/

Gènes et oncogènes: https://www.aurianneor.org/genes-et-oncogenes/

The pill umbrella: https://www.aurianneor.org/the-pill-umbrella-drug-research-went-from-the/

[rakka-kasahara]

2024-11-22

I think the first one had to do with me digging a computer PCB out of the dirt? I remembered it better when I first woke up, but it is pretty much gone now. For some reason, I also got a flashback to an alien or maybe secret facility?

The second also involved a computer. I am at Walmart in my home town in the computer section. Walmart has sort of the older layout for the tech section that they had before I left for college. I am looking at laptops and Brother N and my Mom and Dad are both there. I take a laptop and walk away with it to view the desktop setups. They are where the TVs should be. Most are normal, but a few are funky. There was this all-in-one PC that had a clear window at the bottom so you can see the insides, but the insides didn't make sense. It was way deeper than it could be for such a small computer. Some of the "gamer rigs" on the lower shelf were large squares with glass windows. One even had a sort of printer and game cartridge slot adapter bolted on the front. I got back to the laptop where my parents are and return the laptop I took. However, I noticed I must have messed up the Windows installation because I can't shut it down without it needing to so some sort of repair. I leave and tell my family I am going to check out the clearance section. This was on the other side of the tech section, though it is in a different place in real life. I notice they have some candy, including a tone of dried oranges. I reach up to the top shelf to get some oranges. After looking at the other stuff, they sort of disappear. The other stuff was Halloween things and some sweaters. I grab the sweaters without really looking to see if they are my size. My family is in the clothing section, but it is a little weird in that it is enclosed by walls as it it is its own room in the middle of the building. They are looking at some clothes, and I think I mention I got some clearance stuff. For some reason, I say I got one for my Brother N, and I pull out a Jack Skeleton sweater. It is an extra large and should fit him. That is about when I wake up.

Analysis:

I don't remember much of the first dream, and they may have even been separate dreams. Nonetheless, I think I know the inspiration. I started on a ZX Spectrum clone project the previous night. I also watched the most recent episode of Dandadan, so that might have influenced the other part I couldn't remember.

For the second dream, I actually have an HP all-in-one as a kitchen computer. We played games on those laptops at Walmart a lot as kids when Mom was shopping. For the dried oranges, I guess I am making a sort of fruit bread and am considering buying some candy from the candy store for Christmas, so maybe that is it. I actually got a Jack Skeleton pajama set on clearance last year, so that is defiantly the inspiration behind that sweater. My local Walmart also has a lot of Halloween stuff on clearance.

[helen0086]

Molecular Cloning and Functional Study of the Phycocyanin Cleaving Enzyme CPCS from Spirulina

Abstract: In order to study the catalytic function of algal cyanine lyase CpcS in Spirulina subsalsa, the gene encoding algal cyanine lyase SscpcS was first amplified from the genomic DNA of S. subsalsa FACHB351 by PCR, and then pCDFDuet-SscpcS was constructed as an expression plasmid, which was co-transformed into E. coli BL21 (DE3) together with pETDuet-SscpcB-Ssho1::SspcyA expression plasmid of auxin and pigment synthase. The expression plasmid pCDFDuet-SscpcS was then co-transfected with the cofactor protein and pigment synthase expression plasmid pETDuet-SscpcB-Ssho1::SspcyA into Escherichia coli BL21 (DE3) and recombinantly synthesized into phycocyanin by IPTG (Isopropyl β-D-Thiogalactoside) induction. Sequencing of the PCR products showed that SscpcS was successfully amplified, and the plasmid pCDFDuet-SscpcS was successfully constructed and was able to express the target protein by double digestion and SDS-PAGE.

The cellular product of recombinant phycocyanin PCB-CpcB was dark blue; the purified pigment protein showed a maximum absorption peak at 620 nm and a maximum fluorescence emission peak at 646 nm; the pigment protein was stained with zinc ions, and showed obvious fluorescence under ultraviolet irradiation. In this study, the gene encoding SsCpcS, a cyanobacterial proteolytic cleavage enzyme from Spirulina subsalsa Salina, was successfully cloned, and its product SsCpcS can effectively catalyze the biosynthesis of cyanobacterial proteins. This study lays the foundation for the recombinant synthesis of S. subsalsa cyanobacterial proteins and the development of antioxidant reagents, and also provides a scientific basis for the investigation of the catalytic mechanism of SsCpcS in Spirulina salina.

Spirulina, a cyanobacterium, is widely recognized as an ideal health food. In addition to containing many minerals, carotenoids and other natural pigments, it is also the richest food source of vitamin B12 [1]. Among the pigmented proteins it contains, Phycocyanin (C-PC) has been widely recognized for its strong antioxidant and free radical scavenging ability [2-4], and its significant role in anticancer, antiviral, immunity regulation, and non-toxicity to mammals [5-7]. However, the current extraction process of algal blue protein is complicated, which limits its application. As the biosynthesis mechanism of phycocyanin has been analyzed, the recombinant synthesis of phycocyanin in Escherichia coli using genetic engineering technology is a better choice. It has been demonstrated that this recombinant phycocyanin is physicochemically similar to natural phycocyanin and exhibits good antioxidant activity [8-12]. However, the production of recombinant phycocyanin generally requires the catalysis of lytic enzymes [13-14], so the study of lytic enzymes and their catalytic mechanism is crucial in the heterologous synthesis of phycocyanin.

Studies have shown that phycocyanin has two subunits, α and β [15], and that there are three families of alginate ligases, CpcE/CpcF (E/F type), CpcT (T type) and CpcS (S type) [16-18]. Lysin CpcE/F catalyzes the covalent coupling of the α-subunit (CpcA) of phaeocyanin (PCB)[19] , and lysin CpcS catalyzes the covalent coupling of the β-subunit (CpcB) of PCB[20] , in which PCB is produced by heme oxidase (HO1) and biliverdin reductase (PcyA) using heme as the substrate[21] . It has also been found that some of the S-type ligases need to form a heterodimer with another ligase, CpcU, to perform their catalytic function, e.g., the S-type ligase of Synechocystis sp. PCC6803, which is known as the CpcS-I isoform; however, some of the S-type ligases, such as the S-type ligase of Nostoc sp. PCC7120, which is known as CpcS-III, are able to catalyze alone [22]. However, some CpcS alone can perform the catalytic function, such as the S-type ligase of Nostoc sp. PCC7120, which is called CpcS-III isoform, and CpcS-II, which is only found in marine polychlorophylls, performs the catalytic function together with CpcU-II [22].

It can be seen that the catalytic mechanisms of different types of lytic enzymes are different. Among the commonly cultivated Spirulina, Spirulina salina has the highest total amount of proteins and the widest adaptability to salinity and humidity [23], but there are few studies on the functional mechanism of the cyanobacterial lysin lyase of Spirulina salina. In our previous study, we used CpcS ligase from Cichlidium spp. to catalyze the biosynthesis of the cyanobacterial β-subunit of Leptosphaeria salina [24], and found that, although different sources of cyanobacterial ligases showed similar catalytic functions, their structural differences might lead to different catalytic efficiencies. Therefore, the study of the catalytic function of recombinant Leptospira salina CpcS lysylase may not only improve the efficiency of the catalytic enzyme and increase the production of recombinant algal cyanobacterial proteins, but also help to clarify the taxonomic properties of Leptospira salina CpcS and to investigate the catalytic mechanism of the lysylase CpcS.

Based on the successful construction of the expression plasmid pETDuet-SscpcB-Ssho1::SspcyA for cyanobacterial β-subunit de-coordinating protein and pigment synthase in Spirulina salina [24], the present study was carried out to clone the gene encoding for cyanobacterial proteolytic cleavage enzyme, SscpcS, and co-expressed it with pETDuet-SscpcB-:SspcyA in E. coli by PCR technology. Ssho1::SspcyA in E. coli to synthesize the cyanobacterial protein CpcB-PCB. This study can lay a foundation for the recombinant synthesis of cyanobacterial proteins and the development of antioxidant reagents in Spirulina salina, as well as provide a scientific basis for the investigation of the catalytic mechanism of the cyanobacterial lysylase CpcS in Spirulina salina.

1 Materials and Methods

1.1 Materials and reagents

Spirulina subsalsa FACHB351 was purchased from Institute of Aquatic Biology, CAS. The clone strain Escherichia coli DH5α, expression strain Escherichia coli BL21 (DE3), and expression plasmid pETDuet-SscpcB-Ssho1::SspcyA of phycocyanin β-subunit deacylation and pigment synthase were kept in the laboratory, of which SscpcB, Ssho1, and SspcyA were from Spirulina subsalsa FACHB351, SscpcB, Ssho1, and SspcyA were obtained from Spirulina subsalsa FACHB351. pCDFDuet-1 expression vector was purchased from Novagen; restriction endonucleases Bgl Ⅱ, Xho Ⅰ, and T4 DNA ligase, and the corresponding buffers were from Thermo Fisher Scientific; and the plasmid miniaturization kit, DNA purification and recovery kit, Pfu DNA Polymerase, dNTP, and dNTP were from Thermo Fisher Scientific. DNA  Polymerase、dNTPs ；1000 bp DNA ladder、Protein marker ；The 1000 bp DNA ladder and Protein marker were produced by SMOBIO; Isopropyl β-D- Thiogalactoside (IPTG) was produced by Beijing Boao Toda Technology Company; Ni-NTAAgarose was produced by GE Healthcare. The primer synthesis and gene sequencing were done by Nanjing Kingsley Biotechnology Co.

1.2 Primer design and molecular cloning

The genomic DNA of cyanobacteria was extracted according to the method of Hao Min et al [25]. The polymerase chain reaction (PCR) method was used to amplify the cleavage enzyme encoding gene SscpcS from the genomic DNA of the cyanobacterium Spirulina subsalsa FACHB351, and the primer design was based on the reported sequence of S. subsalsa PCC9445, which was obtained from the NCBI GenBank database (Accession No. WP9445), and the sequence was obtained from the NCBI GenBank database (Accession No. WP9445). Since the CpcS sequence of S. subsalsa FACHB351 has not been reported, the primers were designed based on the reported sequence of S. subsalsa PCC9445, which was obtained from the NCBI GenBank database (Accession No. WP_017305822). The upstream primer for amplification of SscpcS was 5'- GCG AGA TCT GAT GGA CGG TTT AAC ATT TTT TGAG- 3 ', and the downstream primer was 5'-CCTCTC GAGTTACCA ACC ACT AAC AGC ATAAAG- 3 '.

The upstream primer was designed with Bgl Ⅱ and the downstream primer was designed with Xho Ⅰ. The PCR reaction system was 50 μL, containing 0.2 μmol/L primer, 10 ng DNA template, 0.25 mmol/L dNTPs, 1×PCR reaction buffer, and 2U of fast Pfu DNA polymerase (Tiangen, Beijing, China). The PCR program was set as follows: pre-denaturation at 94 ℃ for 3 min, denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 30 s and extension at 72 ℃ for 1 min. The annealing temperature was set from 58 ℃ to 52 ℃, and each temperature was cycled once, and then 52 ℃ was cycled for 30 times, and 72 ℃ was extended for 5 min.

The PCR-amplified target genes were detected by DNA agarose gel electrophoresis and sent to Nanjing Kingsley for sequencing. The amplified SscpcS fragment and expression vector pCDFDuet-1 were double digested by restriction endonuclease Bgl Ⅱ and Xho Ⅰ, then purified and recovered by TAE agarose gel electrophoresis, and then ligated at 16 ℃ overnight under the action of T4 ligase, and then the ligated product, pCDFDuet-SscpcS, was transferred into Escherichia coli DH5α receptor cells and incubated at 37 ℃ in the presence of streptomycin (25 μg/mL). The ligation product pCDFDuet-SscpcS was transferred into Escherichia coli DH5α receptor cells and cultured overnight at 37 ℃ on LB agar plates containing streptomycin (25 μg/mL), and independent single colonies were selected and inoculated into LB liquid medium containing streptomycin (25 μg/mL), and after incubation at 37 ℃, plasmids were extracted by using plasmid mini-extraction kit, and the plasmids were digested by restriction endonuclease Bgl Ⅱ and Xho Ⅰ, and then detected by DNA gel electrophoresis.

1.3 Protein expression and purification

The plasmid pCDFDuet-SscpcS was transferred into E. coli BL21 (DE3) receptor cells by CaCl2 method, and the transformants were screened by streptomycin (25 μg/mL) in LB solid medium. The positive single clones were picked into 5 mL of LB liquid medium containing streptomycin (25 μg/mL), incubated at 37 ℃ until the OD600 was 0.6, and then IPTG was added at a final concentration of 1 mmol/L. The expression was induced by light at 16 ℃ and 150 rpm on a shaking table for 12 h. The supernatant was discarded by centrifugation, and the cells were collected, and then resuspended and washed twice with twice-distilled water, and the cell precipitates were stored at -20 ℃ for spare parts. The cells were collected and resuspended and washed twice with secondary distilled water.

In order to synthesize fluorescent phycocyanin by co-expression of the constructed plasmid pCDFDuet-SscpcS and the expression plasmid pETDuet-SscpcBSp-Ssho1::SspcyA, the two plasmids were co-transfected into E. coli BL21 (DE3) receptor cells by CaCl2 method for in vivo reconstitution, and the transformants were screened with benzylpenicillin (10 μg/mL) and streptomycin (25 μg/mL), and the transformant colonies were picked from solid medium. Ampicillin (10 μg/mL) and streptomycin (25 μg/mL) were used to screen the transformants, and the transformant colonies on solid medium were picked. E. coli BL21 (DE3) containing pETDuet-SscpcB-Ssho1::SspcyA alone was used as the control, and the transformant colonies on solid medium were picked and inoculated into 5 mL of LB liquid medium containing the corresponding antibiotics, and cultured overnight at 37 ℃, and then transferred to 200 mL of TB medium, and then cultured at 37 ℃ until OD600 was 0.6. The cells were collected by centrifugation at 6,500 rpm, resuspended and washed twice with distilled water, and the cells collected by centrifugation were stored for the detection of cytofluorescence or the extraction of recombinant proteins by ultrasonic crushing.

To purify the recombinant protein, 8 mL of pre-cooled Start buffer (20 mmol/L KH2PO4-K2HPO4, 500 mmol/L NaCl, pH 7.2) was added to each of the collected PCB-CpcB cells to resuspend the cell products in the buffer and the cells were broken by sonication in an ice bath. The fractured cells were centrifuged at 12 000 rpm for 40 min at 4 ℃ and the supernatant was collected. The supernatant was purified using a nickel ion affinity chromatography column, and the purified pigmented protein was dialyzed in KPB buffer (50 mmol/L KH2PO4-K2HPO4, 500 mmol/L NaCl, pH 7.2), because the expression vector used was histidine His-tagged, and the recombinant protein was able to bind to the affinity layer medium.

1.4 SDS-PAGE electrophoresis and zinc electrophoresis of pigment proteins

Protein SDS-PAGE samples were prepared by using the pCDFDuet-SscpcS cell product preserved in Experimental Method 1.3. After centrifugation, 45 μL of 1×SDS sample buffer and 5 μL of β-mercaptoethanol were added, and then centrifuged to remove the precipitate in a boiling water bath at 100 ℃ for 5 min. The supernatant obtained was used as the protein samples for SDS-PAGE electrophoresis. After electrophoresis, the sample was stained with Cauloblue and decolorized with decolorizing solution until clear bands appeared on the electrophoresis gel, and then photographed by a gel imager (Geno-Sens 1850, Qinxiang, Shanghai, China) for preservation.

SDS-PAGE electrophoresis of protein samples was prepared by using the recombinant pigment protein PCB-CpcB purified by Experimental Method 1.3, and the purified protein samples of recombinant products without the participation of cleavage enzyme SsCpcS were prepared as the control group at the same time. 50 μL of the purified protein samples were mixed with 45 μL of 2× SDS buffer and 5 μL of β-mercaptoethanol, and the samples were mixed homogeneously, and the supernatant was centrifuged to remove the precipitation for 5 min at 100℃ in a boiling water bath, which was the protein sample for SDS-PAGE electrophoresis. After mixing homogeneously, the samples were subjected to 100 ℃ boiling water bath for 5 min, and the supernatant was centrifuged to remove the precipitation, which was the protein sample, and then subjected to SDS-PAGE electrophoresis. After electrophoresis, the gel was soaked in 1.5 mol/L zinc acetate solution for 15 min, and then put into the gel imager for detection by 280 nm ultraviolet light. After the fluorescent bands were detected, the gel was stained with Cauloblue and decolorized with decolorizing solution, and then imaged by the gel imager and photographed for storage.

1.5 Spectral analysis

The cell products were resuspended in Start buffer, and the fluorescence emission spectra of the expressed E. coli recombinant chromoprotein cell products were examined, and the absorption and fluorescence emission spectra of the purified recombinant chromoprotein were further examined. Absorption spectra were measured by a UV-visible spectrometer (Lambda 325, USA) with a scanning range of 200-800 nm, a scanning speed of 960 nm/min, and a slit width of 1.0 nm; fluorescence emission spectra were analyzed by a fluorescence spectrometer (Perkin Elmer LS55, USA) with a scanning range of 300-800 nm, a scanning speed of 1200 nm/min, and a slit width of 1.0 nm. The fluorescence emission spectra were analyzed by a fluorescence spectrometer (Perkin Elmer LS55, USA).

2 Results and analysis

2.1 PCR amplification and plasmid construction of the gene encoding the lytic enzyme SsCpcS

According to the experimental method 1.2, PCR amplification of SscpcS, the coding gene of lysyl CpcS, was performed, and then the PCR output was inserted into the expression vector pCDFDuet-1 to construct a new plasmid pCDFDuet-SscpcS. The constructed plasmid was double-enzymatically cleaved by Bgl Ⅱ and Xho Ⅰ, and the product of the enzyme cleavage was electrophoresed by DNA agarose gel, and the target gene SscpcS band was found to be clearly visible at 600 bp (Fig. 1), which was basically consistent with the prediction of 588 bp, indicating that the cloning was correct. After DNA agar gel electrophoresis, there was a clear SscpcS band at around 600 bp (Figure 1), which was basically consistent with the predicted 588 bp, indicating that the cloning was correct.

2.2 Sequence analysis of cleavage enzyme SsCpcS

The coding gene of lysin CpcS, SscpcS, was amplified according to Method 1.2, and the sequence of the gene and the amino acid sequence of the protein (Fig. 2) showed that there were 196 amino acids in the protein. Further amino acid sequence clustering of SsCpcS with other cyanobacterial lysin CpcS (Fig. 3) revealed that SsCpcS was distant from NCpcS from Nostoc sp. PCC 7120, and had a high degree of homology with YCpcS from Synechocystis sp. PCC 6803. Since YCpcS has been shown to be a CpcS-I-type cleavage enzyme [26], it was inferred that SsCpcS should also be a CpcS-I-type cleavage enzyme. In order to confirm this assumption, the amino acid sequence of SsCpcS was compared with that of the cyanobacterial lysylase database (http://cyanolyase.genouest.org/), and it was confirmed that SsCpcS is indeed a CpcS-I type lysylase.

2.3 Expression of the cleaving enzyme SsCpcS

To determine the expression product, plasmid pCDFDuet-SscpcS was transferred into Escherichia coli BL21 (DE3). The expression of the ligase SsCpcS was induced by IPTG in E. coli according to Method 1.3, and the expression product was prepared according to Method 1.4, and SDS-PAGE protein gel electrophoresis was carried out. The results showed that there was a clear band around 25 kDa, which was basically consistent with the approximate molecular weight of the protein based on the amino acid sequence (Fig. 2); and there was no band of the same size at the same position in the control group (Fig. 4), which indicated that the constructed plasmid was able to correctly express lysosomal enzyme SsCpcS.

2.4 Function of the cleaving enzyme SsCpcS

In order to study the catalytic function of SsCpcS, the SsCpcS expression plasmid pCDFDuet-SscpcS was co-expressed with the plasmid pETDuet-SscpcB-Ssho1::SspcyA, which is an expression plasmid for the Semi-subunit of Semi-coordinated protein and pigment synthesizing oxidoreductase of Spirulina salina, in Escherichia coli as described in the Experimental Procedure 1.3. The recombinant cells were collected and co-expressed in E. coli. The results showed that the recombinant cells containing pCDFDuet-SscpcS had a dark blue color (Figure 5A inset b), and the fluorescence emission spectra were measured by fluorescence spectrometer, which showed the maximum fluorescence value at 640 nm under the excitation light of 580 nm (Figure 5A), whereas the control without pCDFDuet-SscpcS did not show any obvious color (Figure 5A inset a), and the control without pCDFDuet-SscpcS did not have a clear color (Figure 5A inset b). The control group without pCDFDuet-SscpcS did not show significant color (Figure 5A inset a), and also did not show any fluorescence emission peaks under the excitation light of 580 nm (Figure 5A). These results tentatively suggest that the cleaving enzyme SsCpcS can catalyze the biosynthesis of the fluorescent phycocyanin β subunit (PCB-CpcB).

In order to further verify the catalytic function of the cleaving enzyme SsCpcS and to determine the spectral characteristics of the fluorescent phycocyanin β subunit (PCB-CpcB) synthesized in Escherichia coli, the recombinant product was purified according to the experimental method 1.3, and the absorption and fluorescence emission spectra were examined. The results showed that the maximum absorption and fluorescence emission values of the purified pigment protein PCB-CpcB occurred at 620 nm and 646 nm, respectively, while no absorption and fluorescence peaks appeared in the control group without cleavage enzyme (Figs. 5C and 5D). These characteristics were similar to those reported in a study [24].

In addition, fluorescence quantum yield and molar extinction coefficient are also important parameters for testing biosynthesized pigment proteins [27]. Taking the absorption spectra of natural C-PC proteins and the fluorescence integrals of the corresponding fluorescence spectra of different absorption values as a reference, the fluorescence quantum yield of the recombinant pigment protein PCB-CpcB was calculated to be 0.26 by using the measured absorption and fluorescence emission data; the molar extinction coefficient was calculated to be 5.63×104/(mol-cm) from the ratio of absorbance before and after the denaturation of the pigment protein, based on the denaturation experiment. The molar extinction coefficient of the recombinant pigment protein PCB-CpcB was calculated from the ratio of absorbance before and after denaturation as 5.63×104/(mol-cm). These results indicate that the cleaving enzyme SsCpcS catalyzes the synthesis of the β-subunit of fluorescent phycocyanin in E. coli.

In order to further confirm the covalent coupling between CpcB, the β-subunit deacylated protein of phycocyanin, and PCB, SDS-PAGE protein samples were prepared according to the method 1.4, and protein electrophoresis was carried out. After electrophoresis (Figure 5B), the protein samples were stained with Cauloblue, and it was found that PCB-CpcB and the control without SsCpcS had obvious bands around 20 kDa, which was consistent with the theoretical value of CpcB [28], whereas the cell samples with induced expression of E. coli BL21 did not have bands at the corresponding positions, which indicated that the exogenous product CpcB was obtained in this experiment; After staining with zinc acetate and irradiation with ultraviolet light, a fluorescent band was found in the PCB-CpcB lane, indicating that CpcB covalently binds the phycobiliprotein PCB, which is catalyzed by the cleavage enzyme SsCpcS.

3 Discussion

Spirulina is a widely used health food, and its alginate has significant antioxidant and free radical scavenging properties [29]. Since the direct extraction of phycocyanin from cyanobacteria is a complex and costly process, the production of phycocyanin by genetically engineered E. coli reorganization has a greater advantage [30]. Studies have shown that the synthesis of cyanobacterial proteins in E. coli requires the catalytic action of ligases, such as CpcS, a ligase derived from Arthrospira plantensis FACHB314, which covalently binds phycocyanin to the β-subunit of phycocyanin and synthesizes fluorescent cyanobacterial proteins [20]. In a previous study by this group [24], the de-coordinating protein of the β-subunit of Spirulina salina was successfully cloned and showed some antioxidant potential. Although the pigment-binding fluorescent β-subunit was successfully synthesized by heterologous phycocyanin ligases, homologous ligases may have stronger catalytic ability and enhance the pigment-binding rate.

A number of studies have shown that there is a strong correlation between the pigment binding rate and the antioxidant activity of phycocyanin [24], and the strength of antioxidant capacity of recombinant phycocyanin not only depends on its de-coordinating protein, but also has a strong relationship with its co-chromophore, and the higher the content of the co-chromophore, the higher the antioxidant capacity of the recombinant phycocyanin is [28]. Therefore, in order to understand the function of CpcS, a homologous cyanobacterial proteolytic cleavage enzyme of Spirulina salina, the present study cloned its coding gene SscpcS from Spirulina salina, and used it to catalyze the recombinant pigment fluorescent protein to show its enzyme function, i.e., it successfully catalyzed the covalent coupling of the β-subunit of Spirulina salina cyanobacteria algins and phycobiliproteins in Escherichia coli. The resulting pigmented fluorescent protein PCB-CpcB exhibited maximum fluorescence emission and absorption at 646 nm and 620 nm, which were similar to the spectral characterization of the pigmented fluorescent protein catalyzed by NCpcS cleavage enzyme from Nostoc sp. PCC 7120 that has been reported [26]. This also suggests that, like NCpcS from Nostoc sp. PCC 7120, SsCpcS ligase alone in Spirulina salina is capable of catalyzing the binding of phycocyanin to phycocyanin by the de-coordinating protein of phycocyanin β-subunit.

On the other hand, by analyzing the evolutionary tree of amino acid sequences of the ligase SsCpcS from Spirulina salina and other cyanobacterial ligases, it was found that SsCpcS is a CpcS-I-type ligase. It is generally believed that the participation of CpcU ligases in cyanobacteria is required to form a heterodimer to co-catalyze the binding of CpcB to PCBs, but SsCpcS alone has shown catalytic activity in in vivo recombination, which is similar to that reported for YCpcS and SpCpcS (Spirulina platensis NIES-39) [31,20]. This further suggests that cleavage enzyme CpcS-I alone is also catalytically active, but in the absence of cleavage enzyme CpcU, its catalytic ability may be inferior to that of CpcS-III (e.g., NCpcS), i.e., it may not be able to further increase the degree of pigmentation of phycocyanin, which needs to be further confirmed.

The next step is to clone CpcU, a ligase from Spirulina salina, and to study the pigmentation of cyanobacterial proteins in E. coli through synergistic interaction with SsCpcS, so as to lay the foundation for the development of recombinant recombinant Spirulina salina cyanobacterial proteins with higher antioxidant potentials and more efficient antioxidant agents. In addition, by comparing the catalytic efficiency of SsCpcS with that of other ligases, such as NCpcS, in the biosynthesis of the same algal cyanobacterial protein, we can provide a reference for further understanding of the catalytic mechanism of CpcS, and for the selection of ligases with higher catalytic ability to improve the yield.

4 Conclusion

SscpcS was amplified from the genome of Spirulina salina S. subsalsa FACHB351 by PCR, and according to the amino acid sequence clustering analysis, SscpcS belongs to the CpcS-I type of ligase, which usually forms a heterodimer with the ligase CpcU to perform catalytic action, but also has catalytic function when it exists alone, i.e., it can catalyze the covalent coupling of the β-subunit of the cyanoprotein and phaeocyanin in E. coli, and shows 620 nm covalent coupling between PCB-CpcB and alginin. It can effectively catalyze the covalent coupling of phycocyanin β-subunit and phaeocyanin in Spirulina salina, and it can recombine into the phycocyanin PCB-CpcB in E. coli, and it exhibits the maximum absorbance value of 620 nm and the maximum fluorescence emission value of 646 nm, which indicate that the gene encoding for the phycocyanin lysyl enzyme SsCpcS derived from Spiroplasma salina is cloned successfully in the present study and the product SsCpcS can be expressed in the following ways SsCpcS can effectively catalyze the biosynthesis of cyanobacterial proteins. Therefore, this study can lay a foundation for the recombinant synthesis of S. subsalsa and the development of antioxidant reagents, as well as provide a reference for the understanding of the catalytic mechanism of CpcS in Spirulina salina.

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