In memory of our friend David Johnson

 

David Alfred Johnson Ph.D

2 September 1931 -  4 February 2025

All of us of project GRFT lost a very good friend today

David was one of the project GRFT team from the start.  He specialized in hardware engineering and development. He took charge of particle bombardment technology being used by project GRFT.  He had an interest in this and stated that he thought there were many applications which this technology would be used for in the future and this was only the beginning.  He was inspired by the DIY gene gun that had been built at BioCurious by Jay Hanson.  Based on this design, he built two DIY gene guns for project GRFT. He designed 3D printed nozzles that were optimized for particle spread.  David made many tests with the Gene guns to get the gas pressure adjusted for best cell penetration.  He learned the basics of plant molecular biology in an amazingly short time and helped to get the contamination issues that we were having under control.  

Project GRFT is where it is today, with the micropropagation pipeline in place and gene gun working for transformations, largely because of the knowledge, experience and skills he brought to us.   I personally enjoyed every minute in the lab with David, and I am sure that all of us who had the pleasure of knowing him can say the same.  He was at most of our meetings over the years.  Sometimes he would have trouble with the audio settings on his device, but he would get that working and then have insights to share with us.  

David was thoughtful with the analytical skill set of a great scientist.  He worked very hard and got a lot accomplished. I was always hugely impressed by his abilities to work with a number of different technologies, and to see clearly how they all interact.  Besides being a top scientist and inventor, David had may interests and abilities, for example as an artist and a poet.  David had heart problems.  He said he wasn't in much pain but that he got tired easily.  Little over a week ago during one on the walks that he took for fresh air and exercise, his heart gave out and he collapsed which caused multiple severe injuries.  He was taken to Highland Hospital.  I went to visit him, and he was having trouble breathing. He couldn't swallow or talk.  His wife Leticia was staying with him at the hospital and caring for him tenderly.  I came back the next day, the doctors had changed his meds.  He was breathing without a struggle and looked peaceful.  

      

Ph.D. Physics (1972)

University of Hawaii. Honolulu, HI.

Dr. Johnson worked as a physist at UC Berkeley Lawrence Lab and the Stanford Linear Accelerator Center.

He did much pioneering work with high energy physics.

Dr. Johnson was the founder, director, and president of TiNi Alloy Company where he managed a research team engaged in development of products using shape-memory alloys.

He owns multiple patents and has published papers.

One of his inventions has travelled to the planet Mars

                               David Johnson and Hector Vera

new project GRFT website

 project GRFT presents a sparkling new website

https://projectgrft.org/

We hope you enjoy your time there 

Year of the elusive plasmid

 For the last year, starting in July 2023, we have been attempting to assemble and verify a plasmid to use in the transformation of rice plants.  Two scientists from Johns Hopkins University visited us last July.  They brought the insert containing the gene to express GRFT.  Having no previous experience with plasmid assembly, this task involves a learning curve for me.  There have been multiple fails which are among other things, teachers.   We try not to repeat the same mistakes and like to think we are making progress.     

It's an interesting summer that hasn't left much time for posting on this blog.  We're still alive and kickin'.

  

Early DuPont Gene Gun

When the method of particle bombardment was developed, the first gene guns used a powder charge to propel the DNA coated metal microparticles into the cells of organisms.  Here is a report by the head of gene gun hardware technology, Dr. Johnson on one of these early and now obsolete models of the gene gun.  It's a real piece of gene gun history.  

Gene Gun by DuPont

A.D. Johnson, 5 Mar 2024

This is a first-impression description of the gene gun that was brought to CCL March 3 2024.

It is a very early version of DuPont ‘bioloistics’ devices: it uses a .22-caliber rifle cartridge to generate pressure to accelerate microparticles. The explosion is triggered by an electrical circuit and fires into a chamber through a burst disc. The chamber is evacuated by a vacuum pump through two ports.

   

 

The gene gun consists of two steel enclosures: it is well-engineered and skillfully machined. The left chamber is a pump with (I assume) an exhaust port on the front and two tubes connecting the vacuum pump to the acceleration chamber on the right, closed by a plastic door with O-ring seals. On top of the acceleration chamber is the gun consisting of a cartridge chamber and a box containing the firing mechanism. Mounted on the side of the vacuum pump is a box with a push-button. This box is wired to the

top of the firing mechanism, so I assume it fires a solenoid that extends the firing pin so that it strikes the cartridge and triggers the release of energy.

Cartridge chamber into which is inserted a 22-caliber shell.

Burst-disc holder that is mounted on a shelf in the vacuum chamber

 

The legend says that this is a biolistic DuPont gene gun model BPG

This machine represents an important piece of history. The design was developed by geneticist Dr. John C Sanford working with Edward Wolf and Theodore Klein in 1983 and was purchased from them by DuPont. The history is documented in papers available through Google scholar. Here is a link to a memoir by Dr. Sanford.

http://www.ask-force.org/web/Bt/Sanford-Biolistic-2000.pdf 

Project GRFT is fortunate to have physicist Alfred David Johnson, PhD on board as part of our small but excellent team.  He has built 2 helium powered gene guns for project GRFT to use in the transformation of rice to express griffithsin, a method based on the research of Evangelia Vamvaka, PhD.

Dr. Johnson has solid background in particle physics.  He is the founder, director, and president of TiNi Alloy Company.  With his company he did extensive research in shape-memory alloys for practical applications.  He has several patents on his discoveries (one of which went up to the planet Mars). 

Prior to founding TiNi Alloy Company, Dr. Johnson was employed as a physicist at Lawrence Berkeley Laboratory. Accomplishments include software for bubble chamber experiments of Luis Alvarez and pattern recognition and track reconstruction programs for the magnetic detectors at Stanford Linear Accelerator Center in which the charmed particles were first discovered. As a member of the SLAC-LBL collaboration he coauthored numerous papers in high-energy physics. 

Because of Dr. Johnson's diligent hard work we now have the pipeline in place to transform rice using particle bombardment.  

Deep gratitude!

Project GRFT's 2nd Gene Gun

 David checks the 2nd gene gun that he built for leaks prior to it's departure from CCL to Hector's lab.  If there is any leakage in the tubing or valves, helium, being of low density, will escape.  It is this low density of He, combined with its properties of  being non-reactive, non-toxic, inert and diffusible that make it a good choice as a propellent.  

The first gene gun made used gunpowder as a propellent.  This was crude and dirty compared to today's helium powered gene guns.   Good work, David and Hector.

 

gene gun

When the button is pushed as shown in this photo, a measured burst of helium gas propels microparticles carrying DNA plasmid into the plant cells. The plant cells that take up the DNA are transformed to express a protein.  This method of getting DNA into plant cells is called particle bombardment.  This is one of project GRFT's two gene guns built by physicist David Johnson, PhD.

Project GRFT: How we spend the summer - happiness is a warm gun

 

Our team of volunteers is shooting rice embryos with the gene gun on Saturdays at Counter Culture Labs. 

Sreenivas and Nathaniel from Johns Hopkins University have joined us for the Summer.  They provided the first version of the plasmid containing the griffithsin construct which we have been cloning on Fridays.  The work on Saturday is to get a layer of plasmid to adhere by forming an adsorption ionic bond with 0.7 micron Au microparticles.  This procedure has an element of timing, including vortexing and centrifuging, while keeping everything cool.  Here Anthony prepares a centrifuge.  The rotor is chilled in a freezer before centrifuging.

  First the Au microparticles are washed multiple times with cold ethanol.  The last wash is done with a pH 9.0 borate buffer. 

 

Nathaniel has the pipettes ready with tips and lined up, he will quickly add plasmid, CaCl2, and protamine and vortexed immediately.  

This kept cold while allowing to rest for 10 minutes.  During this time precipitation occurs. 

After centrifuging the supernatant is removed and the precipitate is re-suspended in pure ethanol.

Then the particles are carefully pipetted into the cartridges which will be loaded into the gene gun.  Here David instructs Sreenivas on the cartridge loading. 

The ethanol needs time to evaporate and then the cartridges can be loaded into the gene gun.

Here’s a closer look at the cartridges after loading.

Rice embryos are placed between 2 wire screens in the nozzle of the gene gun.

Sreenivas opens the valve to allow the helium gas to fill the measuring tube of the gene gun before firing.

David furiously takes notes on all of this.

 

 

working with rice in the lab

 After a wet winter, we're glad that the rainy season is over.  

On project GRFT, we are working with rice. 

Some varieties of rice have been used frequently in plant molecular biology because the are easier to transform.  Transformation is a cellular process where the host organism takes up a DNA plasmid. 

Then the host will express the proteins as desired.

In the project we're doing the chosen host organism is rice, Oryza Japonica, var. Nipponbare

.

Here volunteer Lina prepares rice for plating out.

After husking, the rice gets multiple washings of  sterile distilled water, ethanol and a bleach solution.

We have been improving the process and now the rice when plated out is ready for particle bombardment in one week.

Deadly Nipah virus infections are on the increase

"Griffithsin and its synthetictrimeric tandemer (3mG) are under animal trial, showing promising results."

reference from Journal of Biosafety and Biosecurity:

here

Red Cross warning to an unprepared world.

 https://www.aljazeera.com/news/2023/1/30/world-dangerously-unprepared-for-next-pandemic-ifrc

A refresher for those who have been reading these posts or an introduction to those who are new here follows:

GRFT, or Griffithsin is an antiviral lectin. It has shown to inhibit infection by a number of viruses while being itself nontoxic. Project GRFT has a goal of producing GRFT by expression of the gene (derived from a red seaweed) in Oryza (rice).  As rice is a widely grown food crop in many countries of the world, this method has the potential to empower farming communities with limited resources a means to combat viral outbreaks that will undoubtedly occur in the future. We feel that our efforts support global public health, a basic human right.

It should be noted that both Griffithsin and rice are considered nontoxic and safe.

Project GRFT is a group of friends who are volunteering to develop technology to do large scale global biomanufacturing of GRFT through plant molecular biology. 

Project GRFT does not have as its intent to produce pharmaceuticals but rather to provide the raw material for biomanufacturing which we may perhaps call "GRICE"  

As a project of Counter Culture Labs we have non-profit status and we always welcome your donations, both of money and in-kind donations such as supplies (petri dishes, reagents, etc.).  

We are now working in three fully functional laboratories. 

One is located at Counter Culture Labs in Oakland, California

Another is located in Baltimore Maryland at Johns Hopkins University

The newest lab is located near Modesto, California 

This is our effort to respond to the challenge described in the Red Cross warning.  

Project GRFT gene gun

 

project GRFT has set up our first gene gun.  It was designed and built by physicist and inventor David Johnson PhD.  In the video he fires the first shot from the gene gun in the new lab environment.  DNA plasmid coated Au microparticles are shot into a rice callus which will then be incubated at 25C.  Compressed Helium gas provides the force to propel the particles into the plant cells.  

project GRFT does particle bombardment

 Project GRFT, after setting up our gene gun is shooting DNA coated microparticles into rice calluses.

Two of our volunteers, Kat and Patrick, in the hood and washing the Au microparticles with ethanol.

They are using molecular biology grade ethanol and we do 3 washes

after adding the ethanol -

time to vortex!

Rice plate

 

Kat, Patrick and Grier check the plates in the incubator

Every organism has a food they like.  We feed the rice calluses by plating the embryos on Callus Induction Media (CIM) 

They are masses of cells and with care will eventually grow into rice plants 

As they grow we break them apart.

We re-plate the pieces and the pieces grow. 

Callus growth is slow and we are working on monitoring how much growth takes place over time.  

Every organism thrives in a certain temperature range.

The incubator is set at 25C for the calluses.

The calluses on CIM are kept in the dark.  

At later stages different media is used and light is introduced so that photosynthesis can occur and then roots and shoots form.

Another confirmation of the power of GRFT

 "Quite possibly, the most encouraging inhibitor of MERS-CoV is Griffithsin, a 12.7 kDa lectin found in the Griffithsia species (red-green growth). It has three sugar restricting areas that permit it to tie accurately to glycans on CoV protein spikes and forestall viral connection to have cells, with extraordinary strength exhibited in vitro concentrates against MERS-CoV (EC50 of 0.125 M) and various CoV strains (EC50 of 0.00320.33 M) (Millet et al., 2016). Griffithsin additionally seems to have a low foundational poisonousness, with an explicitness list of 303100 against HcoV cells (contrasted with human colorectal adenocarcinoma or fibroblast cell lines) (O’Keefe et al., 2010), demonstrating that it very well may be one of the leading contenders for the animal and clinical preliminaries against SARS-CoV-2 (Mani et al., 2020)."

excerpt from:

In silico investigation and potential therapeutic approaches of natural products for COVID-19: Computer-aided drug design perspective

https://www.frontiersin.org/articles/10.3389/fcimb.2022.929430/full

DNA plasmid under construction

Project GRFT update.  Sreenivas Eadara and the Project GRFT DNA crew in Baltimore are making good progress with the plasmid construct assembly.  

Beautiful bands:

2022 project GRFT ongoing labwork Updates

 

After a rather disastrous Winter with flooding and other problems at CCL, and the closure of one of our other Bay Area labs due to financial limitations, we are recovering. Spring has been a time of growth and progress for project GRFT.  Micropropagation of rice by Hector Vera on agar has got the early problems with contamination under control and cloning is progressing nicely using a progression of media.  We now have plates with happily growing rice calluses that we are continually preparing for transformation by particle bombardment.  Hector is finishing up a lab complete with a gene gun.  UCB students Allison Nakagawara and Maya Douglas joined the project and have been working on preparation of media and buffers, monitoring callus growth and cloning the calluses.  David Alfred Johnson, PhD and David Finn have teamed up to work on the nozzle end of the gene gun. 3d printed parts had ragged edges and were not able to withstand the temperatures in the autoclave.  David Johnson has built two gene guns.  David Finn has located a high-temperature printer to print the parts with clean edges and able to withstand the heat of autoclaving.  In earlier experiments the blast from the gun blew the target plant material completely off the plate.  The solution being tried now is to place the target between to disks of stainless steel fine mesh inserted into the nozzle.  Sreenivas Eadara and the team at Johns Hopkins are working on assembly of the plasmid construct under the guidance of Noam Prywes, PhD (who is doing a research project at UCB to explore rubisco biochemistry and chloroplast transformation technology in plants). 

Photo taken on the 15^th of July 2022 at Counter Culture Labs in Oakland, CA.

From left to right: Allison Nakagawara, Maya Douglas, David Finn, Eddy Spinner, David Johnson, and Hector Vera.

important considerations when preparing projectiles

 Basically when coating particles we need to pay careful attention to:

1. pH: This is most important!
2. Timing of incubation
3. Temperature of incubation
4. Salt concentration
5. Size of particles
6. Freshness of Spermidine (0.1M)
7. 2.5 M CaCl2 is filter sterilized and aliquots prepared ready to use.
8. Purity and concentration of DNA
9. Keeping particles suspended in the buffer solution while adding the spermidine and CaCL2
10. We have seen problems with particles sticking to the 1.5 Ml centrifuge tubes. DNA Lo-Bind tubes should work better.

preparing rice seed to grow embryos and calluses

 Here's Hector in the lab as he prepares rice seed to grow on media plates

https://youtu.be/Qdc9-8sgymg

note: the husked seeds were washed with bleach to sterilize them

Hector's work on project GRFT

Introducing Mr. Hector Vera who is one of our talented, dedicated and hard working volunteers at project GRFT.  Hector speaks from outside a laboratory in Oakland, California where our gene guns are now being built and tested.  

Open Insulin - changing the paradigm

 The older sibling of Project GRFT is Open Insulin. They recently made a quality introductory video about their work which may be viewed here    We share the goals of  medicine for people, not profit, and valuing open source technology.