Quote from Greta Thunberg

 

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!