Hello All!

I guess my journey with Laccase enzyme has ended this week 🙁 The visiting scholar is so busy with her research and she is facing some issues with her project as well and I don’t want to bother her any longer.  I am hoping that before she leaves for Spain, I can continue with what we have started.

The following were the highlights this week;

a)  Dr. Yu showed me how to perform the Drop Spot Test.  He needs to do this to confirm the efficiency of the phages (in different dilutions) we have been using and check the condition of the bacterial stock as well since we have been having not so good results in our efficiency test.  We found out from this test that the bacteria we’re using has been contaminated with phages.  The test also revealed that there was so much media in the solution, so there is a need to purify the phage as well.

b)  Dr. Chu showed me the process of purifying the phage solution.

c) I was taught how to harvest/isolate and purify phages of interest from a plate.  I think this is the best take away for the week.  I have been wondering how to isolate the phage from the culture.  After isolating the phage, we did the efficiency test for the phage to determine its polyvalency.  It’s amazing that these phages come from one source and grew in the same culture plate yet they can evolve so fast.

d)  We also did a growth curve for the bacteria used in the efficiency test to determine it’s growth over 14 hours  using the plate reader.

Dr. Yu asked me to do more literature review on colorimetric detection of different bacterial host.  This is the direction of my project.  I was able to find some interesting articles but I am not sure If we can conduct the experimentation in the lab considering the time left. This is a big project and they haven’t started it so far.  I am contributing to the project by reading articles on how to do it.  I feel good about it although it is a pain searching and reading articles but my mentor is grateful for all my efforts 🙂

I am feeling the pressure already.  It seems like time flies so fast especially if there’s a lot of things that you want to accomplish.  However, I am taking it one day at a time and enjoying every moment of the experience! Till then….

Greetings everyone!

My 2nd week was not as long as the 1st week but it’s still packed with a lot of information.  Let me start with the applications on these polyvalent phages to water treatment if in case you are wondering.

Applications

a)  These viruses (polyvalent phages) can target bacteria that have shown resistance to antibiotics.  This type of biological control could be used in remediating problems in the water such as bacteria-induced corrosion of pipes.

b)  Conjugated or immobilized polyvalent phages could be used to effectively infect problematic bacteria in a biofilm.  The immobilization could be engineered (e.g. using magnetic nanoparticles) to enhance the delivery system and target these problematic bacteria.

The phages studied in the lab have been tested on the following bacterial hosts; Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella typhimurium.   These bacteria are known to be prevalent in wastewater.  I am thinking of using the E. coli for the lesson that I have thought last week.   The results of the different investigations about polyvalent phages can lead to a system where we don’t need to use antibiotics or chemicals anymore in treating wastewater.  So it is really a promising endeavor!

In the lab, we continued the experimentation last week using the different dilutions of phages and bacteria to find a good result of viral plaques for isolation.  The 50 uL (undiluted) and 100 uL of phage showed the best result so far.   Dr. Yu suggested to also try 100 uL and 200 uL of bacteria to create a better bacterial lawn and compare the results.

I also happened to have a short chat with another visiting scholar from Brazil since her project is also on polyvalent phages.  She mentioned the method called “Drop Spot Test” where it works like the antibiotic test on bacteria where you can see a circular zone of inhibition.  In this test, you can drop a 10 uL of phage solution and see the plaque formation after 24 hours.  I think it was such a very cool idea for a lesson.  Although this is just a qualitative test but students can really see effects of viral lytic infection.   Productive conversations to different people can really bring a lot of valuable information most of the time! I just need to try this myself using resources that are available in the classroom (e.g nutrient agar and phage and bacteria purchased from vendors like Carolina Biological and others)  and see if it works.

As for the enzyme laccase, since we finished the immobilization last week, we wanted to find out if the sample has been completely immobilized by measuring its activity using the plate reader.  Once this done, we can proceed with the testing of the effectivity of the enzyme when it comes to its ability to oxidize the methyl green dye, a representative of the dyes that are commonly found in wastewater from the textile industry.  And oh, since the nanoparticles used to immobilized the enzyme is magnetic (Silica-coated magnetic nanoparticles), we don’t need to centrifuge the sample to obtain the supernatant fluid, we just used a Neodymium single disc magnet to bring the enzyme down to the bottom of the eppendorf tube, pretty cool huh! 🙂

I will never forget this part of the experiment because I did something wrong to Yolanda’s magnet (huhuhu!) Don’t ever underestimate the power of the neodymium magnets.  I accidentally placed it  at around 6 to 8 inches away from another magnet that is thicker and oh boy, the disc magnet was drawn to the bigger one and it cut into half.  I felt so guilty that I felt like I have to replaced it so I went online, searched for it on amazon and ordered one 🙂  Charge to experience.  You learn as you go! No regrets though 🙂  Stay tune for week 3!

Hello All!

I was assigned into Dr. Alvarez’s lab under the mentorship of Dr. Pingfeng Yu to work on Poyvalent Phages with the assistance of Dr. Chu, a visiting scholar from China whose expertise is also on polyvalent phages.  I could go to either of them depending on who is available in the lab.  I was also introduced to different researchers and visiting scholars and had a chance to speak to them about their projects.    In addition to polyvalent phages, I got interested with the work of Yolanda Moldes, a visiting scholar from Spain because she is working on remediating emerging pollutants in wastewater using the enzyme laccase.  This really sparked my interest because I want to know how enzymes work in removing pollutants in the water such as dyes, BPAs and other compounds that are known to be endocrine disruptors. I asked her if she could show me the process of the investigation and graciously said ‘yes’ to my request but my primary investigation is on polyvalent phages.

Viruses that infect bacteria are called phages or bacteriophages.  Usually, a phage is specific to its host, however there are times when one phage could infect multiple strains of bacteria (hosts) hence they are called polyvalent phages. The phages used in the lab were isolated from a wastewater treatment facility in Houston.  The isolation of the phage(s) is not part of my investigation, instead my task is to characterize these phages as they infect a certain host by understanding the compounds released by each species of bacteria as they lysed due to viral infection.  What these compounds are and how we can detect them are the challenges that I need to face throughout the course of the investigation with the assistance of my mentors.

This week, I learned the following;

a)  inoculating bacteria

b)  culturing phages (I learned that you need two layers of agar, a fixed agar and a soft agar on top where bacteria grow and create a bacterial lawn.  The agar used was LB. The phage or phages will infect these bacteria and form viral plaques – zone of infected cells which can be visibly seen as clear circular structures in the bacterial lawn).  This part of the lab is an initial idea of a lesson that I could write to show lytic infection of viruses since I haven’t come across a lab on lytic infection so far.

c) diluting the concentration of bacteria and phages using the 10-fold serial dilution method.  A good review for me since I don’t really do this much in my own lab.

d)  perfecting my pipetting skills (Pipettors of different volumes are just one of my best buddies in the lab.  I did a lot of pipetting throughout the week that I think I’m becoming like a pro on this! lol!)

The goal of the first week is to determine the best phage to bacteria ratio which could produce good size of viral plaques where we can isolate a phage for purification.  This wasn’t attained the first day so we kept modifying our volumes and dilutions throughout the week.

e) immobilizing the enzyme laccase using magnetic nanoparticles (with Yolanda’s assistance, I was able to observe the process of immobilizing enzyme using the nanoparticles.  It was a complicated process which I think can’t be done in my school lab, but I will continue to explore and maybe bring this investigation in my classroom.  Several researches showed that enzyme immobilization is more stable and more effective in catalyzing reactions than those free ones. Now, I am thinking if there is a simpler way to immobilize this enzyme but still using nanoparticles that can be done in the classroom lab, my hopes are high!)

It feels like my week was really packed.  I had plenty of take aways and I love every moment of it.This is why lab immersion stands out as an avenue for professional growth because you experience the process itself including failures.  I am excited of the remaining weeks to come!!!