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

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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.