New Phase of My Project

In the beginning of this week, I started to transform my bacteria (E.Coli and Agrobacteria) again because I was not able to transform my Agrobacteria last week. On Monday, I did transform bacteria and follow the same procedures as I did last week which I couldn’t transform to Agrobacteria again. On Tuesday, Josh told me to transform my Agrobacteria again, but this time I should place my microtubes which contained the + pGLO and - pGLO tubes to a 95 degree C water bath for exactly 10 minutes instead of 42 degree C water bath for exactly 50 seconds. Then, I returned the tubes to a container of crushed ice and cultured my bacteria in agars, to later incubate them ina temperature of 37 degree C. On Wednesday, the first thing I did was to observe my Agrobacteria which I cultured on Tuesday and Monday. When I checked my agars from Monday with a UV light, nothing glowed, but transforming of E.coli was successful and it glowed and I exposed my petri dishes. The worst part is nothing did grow in my agars when I transformed them on Tuesday, but I put them back to the incubator. Today, after almost 48 hours I check my petri dishes. Unfortunately, petri dishes were clear and nothing did grow except that I saw two tiny colonies of bacteria on - pGLO LB/amp agar and I decided to keep it to grow more bacteria because I want to identify it with gram staining method next week. 

 According to the evidence, probably when I put my microtubes to a 95 degree C water bath for exactly 10 minutes. I did kill all of my Agrobacteria but we (Josh and I) decided to put some Agrobacteria to a 95 degree C water bath for 10 minutes again. Then, extract DNA to see that any Agrobacteria remain. In this way I’ll learn how to extract DNA and with the result of Bacterial DNA Extraction, we can tell why Agrobacteria didn’t grow. 

The good thing that happened today was the Vibrio Fischeri bacteria that josh ordered for my project arrived. I was so excited and started working with bioluminescence bacteria right away and leave DNA Extraction for next week. 

 

At first, I checked my Vibrio Fischeri tube in total darkness for 10 minutes to observe bioluminescence. Then, I started to transfer the culture that I did in the Biological cabinet, because this culturing should be done in a disinfected place. I cultured Vibrio (photobacterium) Fischeri into the two photobscterium agars that the company sent to us, and one luminescent agar which I made two months ago. At the end, I placed my tubes in the incubator at 23 degree Celsius.

Bacterial Transformation

 I tried to transform Agrobacteria and Escherichia coli (E. coli) with plasmid DNA . Bacterial Transformation is the “process by which the genetic material carried by an individual cell is altered by the incorporation of foreign (exogenous) DNA into its genome” (MedicineNet.com, “Definition of Genetic transformation”).

At first, I used a micropipette to transfer 250 ul of transformation solution (CaCl₂) to the tube labeled + pGLO and another 250 ul to the tube labeled - pGLO. Then, I placed the tubes in a container of crushed ice.

I used a sterile loop to pick up several colonies of my bacteria (Agrobacteria and E.Coli) from the starter plates and transferred the bacteria to the + pGLO tube by spinning the loop rapidly after it is immersed in the liquid. I repeated this procedure by transferring several colonies of bacteria to the - pGLO tube.

I immersed a sterile loop into the bottle containing plasmid DNA. When the center of the loop was coated with a soap-like film, transferred it the + pGLO microtube .The - pGLO microtube will not receive any plasmid DNA. Then, I placed the tubes back tubes in a container of crushed ice for 10 minutes.

While I was waiting for the tubes to cool, I labeled my agar plates with a letter that indicates the type of bacteria that they will receive.

Plate	Label
LB/amp	+ pGLO
LB/amp/ara	+ pGLO
LB/amp	- pGLO
LB	- pGLO

I placed the foam microtube holder containing the + pGLO and - pGLO tubes to a 42 degree C water bath for exactly 50 seconds then return the tubes to a container of crushed ice. Tubes should stay in the ice for 2 minutes.

After 2 minutes, I removed microtubes holder from the ice and place them to the foam microtube holder. Then, I added 250 ul of LB broth to each of the bacterial cultures (the + pGLO tube and the - pGLO) with a micropipette and tubes were incubated at room temperature for 10 minutes.

I tapped the closed tubes with finger to mix. Then, I used using a micropipette to transfer 100 ul of the solution from the + pGLO to the surface of the agar in the plates which were labeled +pGLO. A different pipette tips were used to transfer 100 ul of solution from the - pGLO to the surface of the agar in the plates labeled -pGLO. 

I used different sterile loops to spread the mixture over the entire surface of the each agar by quickly skating it back and forth across the plate surface. Plates were stacked and tapped together. I placed the stacked upside down in 37º C incubator for 24 hours.

I checked my plates after 24 hours with UV light and just E. coli witch was cultured on LB/amp/ara plate has been transformed.