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.

New Clinical Trials of Griffithsin pharmaceutical designed against infection by coronaviruses

This is huge.  

Dr. Kenneth Palmer's group have been working hard to get a Griffithsin based pharmaceutical into clinical trials for some time and now a phase 1b trial is beginning. 

The pharmaceutical is designed to prevent entry into the cells by coronaviruses.

https://clinicaltrials.gov/ct2/show/NCT05437029

https://discoveria.com/studies/NCT05122260

The Pharmaceutical is in the form of a nasal spray which is an effective route to block the coronaviruses that gain access through the respiratory system.

Phase one trials are the first of four phases of clinical trials with human volunteers (They are now accepting volunteers for this study) designed to test the pharmaceutical on healthy individuals to see if any unwanted detrimental effects occur from the drug.  

If and when Q-Griffithsin is approved by the FDA, the method of biomanufacturing will likely involve large scale expression in plants, in this case nicotiana (tobacco).  

Congratulations, Dr. Palmer, on getting clinical trials underway!

Free STEM supplies for educators

 Our friends at Bio-link Depot in Oakland, California are having an event 1 May '22

Registration is required please follow this link:

https://mailchi.mp/dc53af3df94b/update-your-contact-info-save-the-date-for-open-house-8658925?e=d718c48211

https://www.eventbrite.com/e/bio-link-depot-open-house-may-1-tickets-320360004397

First phase 1 clinical study of GRFT for prevention of HIV

 Researchers at Albert Einstein College of Medicine completed phase one clinical study of a griffithsin topical.  This is designed to be applied as a gel to the vagina before sex to inhibit HIV infection.  Phase one trials are done are to see what effect the pharmaceutical has on healthy humans.   The results of this study are very promising,  This is a huge step forward, and we look forward to the next phases of clinical trials.

Here's a link to the paper:

 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0261775 

Meanwhile our group is looking into what the optimal pH of a buffer for adhesion of DNA to W and AU microparticles would be.   We would like to hear from anyone who has done particle bombardment on the subject of what buffers worked best.

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 particles - theory

 

What is happening when we coat either Au or W particles?   

Adsorption

Adsorption is where a layer of molecules cling to a surface.

1.       There is the adsorbate (i.e. DNA plasmid)

2.       There is the adsorbent (i.e. W)

What attracts the adsorbate and the adsorbent to each other so that they are held together in Adsorption?

DNA has a – charge.  W has + charge.  Under the right conditions, a bond is formed.  If the DNA is in solution with the W and the bond forms, the DNA will precipitate and adsorption occurs.  The DNA is not present in the solution anymore, it is now coating the W microparticle.

That’s very beautiful and simple to understand but as may be expected the devil’s in the details.