Caution Failed Attempt

Figure 1. The duck tape around the rubber line.

Figure 2. "Caution: The floor is wet!"

Figure 3. The papers are all wet.

I have worked on the gene gun this week and had no success. But I only fall to get back up again and try. I was missing some threaded tape to seal the threads between the metal for a more pressurized seal. I was trying to reach a goal of 80psi but only got to 60psi before the rubber line ruptured and leaked the air. I have modified the gene gun with some duck tape because of my lack of finances in my project. I have also posted a pic of a joke below the modified gene gun. Can anybody tell me if they get the joke.

Dung Cannon

Figure 1. This fungus is a close up of this 2-4cm tall stem with somewhat a black top hat full of spores. The vesical below fills with fluid creating a pressure to shootoff the black ball spore like object above.

What would most of my fellow inters think was the fastest accelerating object on earth? A land animal like a cheetah, a hemi 357 engine with aftermarket add-ons for better performance, a jet in the sky, or a fungus. The answer is a fungus known as Pilobolus crystallinus. A fecal microorganism that cycles thru the animal grazer intestinal system back out into fecal matter (feces) is part of their life cycle. The question is how does this fungus get back to the mouth or entryway of the digestion system? This 2-4 cm tall fungus shoots its spores about 6feet away from its home of feces to a clearance where grazers eat. This 45mph shot in one millimeter is the fastest accelerating object on earth. I have included a video and a picture of the fungus below and above.

Species Education

Figure 1, 2, & 3. The pictures from the Wildlife World Zoo flamingo, aquarium with sea turtle (absent), and vulture. (from left to right, respectively.)

The trip to the Wildlife World Zoo was a lot of fun from touching the stingray to their awesome aquarium. The view of the animals was better than the Phoenix Zoo in my opinion. Although Amanda, Josh, and me observed a vulture watching something move in the cluster of leaves and shrub below its feet outside of its cage, we believed the mice were diverting the attention away while another grab some its fruit inside the cage. Moreover, I wished we had more time to experience the nursery they had. I look forward to the next fieldtrip to the brewery. I have uploaded a video of a snake devouring a crocodile. The video is graphic in some sense.

Simple Gene Gun

Figure 1. The blue box contain 13 mm in diameter disk filters placed atop of the pre-filter shown above in the lower left corner of the close up of a Swinnex filter. (Note: the disk filter 3 microns of material passing thru.)

I worked on putting a gene gun together this week with a 16-piece compressor set from Home Depot and a used foot pump. The development of this very simple and inexpensive gene gun seems it might work. On the other hand, I may have come across some problematic obstacles heading down the road of this future apparatus. For instance, the Swinnex chamber with the pre-filter in it has an inlet capacity of 50psi or 3.5bar. The maximum pressure I will need for the successful acceleration of the tungsten particle coated with the desired genes to shoot thru this filter is about 80psi or 5.5bar. Figure 1 above show the device separated into its individual pieces. The red arrow indicates the flow of the air being compressed. I, also, added a pic of the Swinnex filter in the lower right corner of Figure 1.

The attaching of the coupler and other threaded areas needed Teflon tape for proper seal. I tried to use rubber tape that was lying around in the robotics lab room.

All This Reading for Nothing

Diagram1. This poor diagram shows the mechanism of how helium used an force to push the particles of gold coated with the desired DNA in the syringe filter shoots into target of plant tissue. The purple oval shaped circles above the box is where the solenoid would go and the other two is the pressure gauges within the  box.

I started reading an article I found about developing a simple gene gun apparatuses. It included welding pieces of 0.5 centimeters (cm) thick stainless steel sheets into a cube like shape. The open side was covered with 1.5cm thick Plexiglas with 1.0cm thick rubber gasket around the edges. The thick Plexiglas would serve as a door to the cube. Thus, the overall specifications would have a solenoid attached to the top which it served as the mechanism of the gun. Diagram 1 above shows the specifications of the simple gene gun. The vacuum served to silent the nose of the compressed helium shooting thru the shaft of the micro tube of the solenoid. The coated gold particles with the desired genes are shot at a very high acceleration to intervene with the nucleus of the plant cell without disrupting its normal function. In conclusion, the desired DNA is inserted into the DNA of the plant DNA if it was cut in some random area. This will overall make the DNA code for some kind of gene expression.  

Josh on the other hand had a cheaper and less efficient way of making a gene gun with a super soaker. Same concept but cheaper parts and instead of gold for its particle to be coated it will be the chemical powder tungsten. Also, the gas helium would be switched out for nitrogen and carbon dioxide powder.

I Can See Memory

The process of how memory is formed is visually caught in real time for a couple of seconds. The mouse used as an in vivo experiment for this accomplishment had its mRNA or messenger ribonucleic acid tagged with green fluorescent chemicals. Messenger RNA encodes for the beta protein that shapes and structures the brain cell for memory storage. The part of the brain known as the hippocampus was stimulated to watch the mRNA go into process. The hippocampus role is separating events and experience of short term memory to long term memory. Thus, driving a car for the first time creates paths by the mRNA by encoding the nucleus of the neurons to create molecules that shape the path of the dendrites synaptic communication between other neurons.

Figure 1. The process of how the mouse mRNA was tagged with fluorescent green (A). The molecules forming for roles of creating memory of the neuron out to the dendrites (B, C).

Park et al. (2014). Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science. 343 (6169): 422-424. doi: 10.1126/science.1239200

Native Instincts Kicked In

Well I can say I have a green thumb. The pea plant seeds came out with all pots successfully growing into sprouts. Figure 1 below shows the 100% success of all pots growing pea plants. Furthermore, the green tray was split with my lab made divider using yellow tape and cotton balls. I split the tray so the plants wouldn’t share the liquid of DI water and faucet water, but it turned out that both liquids different in pH both grew the plants. I think my last plants didn’t grow because the temperature was at 34 degrees Celsius; also, the soil was soaked rather than lightly moisten and the mixed soil with vermiculite.

Figure 1. The bottom left corner shows a close up of the success. There is a yellow arrow showing the yellow tape dividing the two rows of pots.

I, also, sampled two locations off campus for agrobacterium tumefaciens (A.tumefaciens). Figure 2 below show the samples in 0.1% Peptone of 45mL with the corresponding test tubes. Once the sample was chiseled out with sprayed 70% alcohol of a hammer, flat head screwdriver, and chisel, it was immediately put into a urine sample cup of Peptone of 90mL for isolation of bacteria. The piece of the crown gall was then rinsed with sterile DI water then cut using a sterile scalpel and tweezers. This whole procedure was performed in the flow hood for low risk of contamination. The pieces cut were place in two sterile test tubes of 6mL of Peptone and two sterile test tubes of 6mL of Tryptic Soy Broth (TSB). Then, the eight test tubes were stored in a cabinet for room temperature of 25 degree Celsius for proper growth of A.tumefaciens.

Figure 2. The neon green top rows of test tubes are tree "A." The next 2 bottom yellow rows are tree "B." Basically, the top row has TSB. The next row has Peptone, then the next row is TSB. The final row has Peptone. 

Interesting WEEK,

In the beginning of this week I started mixing my own soil for perfect growing conditions for my pea plants. The taxonomic name would be Pisum sativum (P. sativum). These dicot plants would be a good host for infection of the Agrobacterium tumefaciens (A. tumefaciens).  Figure 1 below show the arrangement of the seeds in the pots, and I used 70/30 of soil/vermiculite with a pH of 6.23. I was, also surprised on how to calibrate a pH meter. Furthermore, Josh told me that there was a pH meter for the soil that Matt had in his room. This meter gave a reading of 7 and the calibrated meter gave a reading of 6.23. On the other hand, the soil needed to be slightly acidic for growing conditions. Thus, the top row would have only DI water and the bottom row would have regular faucet water. I, also, trained newbie intern Alex on my procedure to prepare media and sampling technique used to obtain bacteria on the made contact plates with TSA.

Figure 1. The pots are set up just the same way as this previous pic, but there is a divider in the middle so the water I put in don't mix.

Failed Experiment

Figure 1. This is just five of the twenty randomly used for observation and statistical analysis of the CFU. The rate of this group is fair.

Figure 2. The red obscure circular shaped colony in the top middle row is very interesting. The small white of a guess would be S. epidermis and the yellow medium sized circular would be S. aureus. The rate would be poor.

Figure 3. I would say the rate for this group would be fair.

Figure 4. 

Figure 5. I would rate this group as very poor. There is a rigid circular shaped colony in the bottom row of the left plate.

Figure 6. I would rate this group as poor.

The plants that I seeded with Sonora wheat and Anasazi beans died because there was no watering due to my absence of the lab. Although I infected three Anasazi beans with the possibly A. tumefaciens, I wasn’t able to get any results because of my poor responsibilities in the lab. On the other hand, my first project with the fomites is going somewhat okay. I have sampled more than 8 locations around campus, and each location had about 20 plates used for each site. If my calculations are correct, I used about hundred and eighty plates, and trashed about a sleeve of incorrect plates when pouring. The figures above show some of the randomly obtained samples from each site that was going to be analyzed using Photoshop software. Furthermore, I would rate the group as poor, fair, or acceptable for either necessary action or non-necessary action to be handled in the caption.