S3 27: See Ya Next Semester

See Ya Next Semester

This past week, I focused on testing another an alternative X-gal assay on yeast cells. This was the second alternative procedure that I had tried and it was successful. I had some extra time in the lab before the semester comes to a close, so my research mentor suggested that I try transforming the other two DNA samples again. The rationale for attempting a second transformation was because they previously tested positive for LacZ. Going back to the DNA sample that we were able to extract, I performed a miniprep on the bacteria cells to increase our plasmid yield. Hopefully, I will be able to have a successful DNA plasmid extraction from the other two samples and all three DNA samples can be sent for sequencing.

S3 26: Rounding Out the Semester

Rounding Out the Semester

I can believe the semester is almost over in less than three weeks. I'm a few steps away from getting my DNA plasmid sent out for sequencing. I had a hiccup in the lab last Wednesday. It turns out I used the wrong Transformation Protocol when transforming my DNA plasmids into E-coli cells. This mistake was definitely a reminder to follow my gut and ask questions when I'm not 100% sure of something. I should have known I was making a mistake when I was confused while reading the Yeast Transformation protocol. Luckily the correct protocol was only three simple steps, and I was able to redo the transformation. After the cells were transformed with the plasmid, we replicated the cell colonies onto M63 plates. The purpose of replicating the cell colonies onto the M63 plates is to isolate the prey plasmid from the bait. The colonies that grew on the M63 plates were then re-streaked onto an LB Amp plate to ensure that we will have a sufficient amount of the prey plasmid to be sequenced. Next week I will perform a miniprep on the cell colonies that grew on the LB Amp plate and then they will be ready for sequencing just as the semester comes to a close.

S3 25: Transformation of DNA into E-coli

Transformation of DNA into E-coli

After the X-Gal Assay was performed and we saw the presence of Lac Z, we isolated the DNA from three colonies by performing a Zymoprep for a liquid culture. After the zymoprep was complete, we tested the purity of DNA before we transformed it into E-coli cells. The purpose of transforming the DNA into E-coli cells is to try and isolate the prey from the bait. The transformed cells will be left to incubate for 4 days on LB Amp plates containing Leucine.

S3 24: XGal Assay

XGal Assay

Last week ended with a restreak of yeast colonies onto master plates with minus Tryptophan, Leucine, Histidine with 50mM of 3AT. After letting the plates grow for about a week, we were able to perform an assay on 3 colonies. The assay results revealed that the yeast cells expressed the β-galactosidase enzyme. When X-Gal adheres to the β-galactosidase, a blue pigment will be visible. This activity indicated that there is a LacZ gene present. In order to verify the results, the three samples will be sent out for sequencing. My favorite part about this assay is that it requires the use of Liquid Nitrogen, and let's just say that it was a very fun day of "science-ing" in the lab!

S3 23: Yeast Transformation

Yeast Transformation 

This week in the lab, we successfully transformed the Bait and Prey on minus Tryptophan, Leucine, and Histidine plates with 50mM of 3AT. After a growth period of about a week, each plate was analyzed and sixty-five colonies were restreaked onto minus Tryptophan, Leucine, and Histidine plates with 50mM of 3AT. The plates will be given a growth period of another week. The colonies that are able to grow on these plates will then be assayed and then sent for DNA sequencing. 

S3 22: Mellow Jello

Mellow Jello
This past week in the lab was pretty mellow. I came into the lab on Monday to take yeast cells that were backdiluated from the previous night and transform them on agar media that included the 3AT. I performed a total of 31 transformations (30 samples and 1 control). The transformation takes about 2-4 days to see any results.I will head back to ASU at the end of the week to check the results.  Additionally, I am working with Dr. Marshall to create a website  with the goal of having the Y2H Transformation protocol and supplemental protocols available so that students from other college campuses like Phoenix College, South Mountain College, and Glendale College can learn about Y2H research and replicate the experiments.

S3:21 Whelp, that's Research.

Whelp, that's Research.....
Whelp, the replica plating didn't turn out as we expected, so we decided to just perform a transformation on a media plate with the 3AT already included. After, I made a new batch of agar media, an overnight culture of yeast cells was started. The next morning, we noticed that the cells weren't growing at the needed rate. We think the cells weren't really growing because of the age of the original yeast culture. Then, it was decided to go back and redo the bait transformation, but I am okay with that because it means more practice for me. The week ended with the bait transformation on a -Tryp media plate. If the transformation is successful, we will see cell growth on the -Tryp plate, and then start an overnight culture in preparation of transforming the cells with the DNA library.

S3:20 Replica Plating

Replica Plating
This week in the lab, I learned a new lab skill called replica plating. Replica plating involves velvet sheets and an apparatus to hold the velvet sheet in place during the replication. Essentially, you take the colonized plate that you want to replica and lightly press it over the velvet and then you take the new plate and lightly place it over the colonies on the velvet sheet, giving you an exact replica of the colonies on a different plate. The purpose of this replica plating was to regrow our colonies on media plates that had 3AT on it.

S3:19 That Yeast is Posionedddd

That Yeast is Posionedddd
This week in the lab, we moved onto the step where we were going to "poison" our yeast cells. When we initially did the first transformation, we noticed that large colonies were forming on the plates which indicated that the bait was still causing transcription and producing histidine. With that observation, we decided that we needed to figure out which concentration of 3AT we could get our cells to grow in without the bait causing the histidine production during transcription. With that said, I made five different concentrated solutions of 3AT and made sample transformations to see which concentration will work best with our needs. Hopefully, the next time I go into the lab, I will be able to see which concentration worked.

S3: 18 Transformation Tuesday

Transformation Tuesday

We performed the yeast transformation of the bait and followed the procedure listed below. The yeast transformation was successful. After the yeast cells were transformed with the bait, we noticed that histidine was still present, so we decided to prepare the cells to be grown on media plates with 3AT. So far in the lab, I have made the media with five different 3AT concentrations. After the media is ready, I will transfer the yeast cells to the media. We will be looking for colonies that are able to produce higher levels of histidine. 

Transformation Procedure: 

For one transformation micro-centrifuge 500mL of competent cells at 500xg for 4

minutes.

Add 0.5mL in EZ 1 solution to wash the pellet. Vortex the cells and discard the supernatant.

Add 50uL of EZ 2 solution and vortex the pellet.

Mix 50uL of competent cells with 3uL of DNA (Bait) and add 500uL of EZ 3 solution and mix.

Incubate samples at 30˚C for 45 minutes.

Sterilize an L-shaped spreader by dipping in ethanol and lighting it on fire but be careful not to heat the spreader.

With an L-shape spreader, spread all the sample on yeast media plate that contains  the –tryp/-Leu

Incubate the plate at 30˚C for 2-4 days so the transformed yeast cells can grow.

S3:17 Hickup in the Lab

https://today.anl.gov/2016/08/safety

Hickup in the Lab
This week in the lab I had a minor setback. On Sunday night a back dilution for the yeast culture was started in preparation for the transformation phase of the project. On Monday morning, when the yeast cells were checked for their phase of growth, it was realized that they were not in the optimal growth phase. After, we decided that it was best to redo the back dilution of cells to make sure they were in their optimal phase of growth before we performed the thirty-five transformations.  I'm looking forward to performing the transformations next week.

S3:16 Transformation Tuesday

Transformation Tuesday

This week in the lab, we took the L40 bait and transformed the cells onto media plates. The cells grew over the course of a few days. Before we transformed the bait onto the plates, I had to prepare the media, autoclave it, and then pour the plates. It is a fun process but can get a bit messy because the agar turns into a jello-like consistency pretty quickly once it gets spilled. Next week in the lab, we will work on the next steps in the transformation process and do a total of thirty transformations. 

S3:15 Falling Back into the Rhythm of Science-ing

First I must express that I am glad the Fall semester has begun. During this semester, I will be at ASU-West under the direction of Dr. Marshall. The goal for the next few months is to become familiar with the Yeast Two Hybrid Protocol. Additionally, I will be experimenting with different Yeast Two Hybrid Library screens. The video above depicts a segment from the DNA Plasmid Maxiprep that we performed this week in the lab. It was as slimy and slippery to work with as it looks in the video! Next week, we will work on transforming the bait. I can't wait!

S2:14 Another Semester in the Books

Another Semester in the Books

The last week of the internship always brings bittersweet feelings. On one end, we are happy to be wrapping up our projects and presentations but on the other end we are preparing to part ways with some of our fellow team members. My favorite part of the semester has been traveling to Vegas with fellow teammates. I learned of another conference happening in DC this fall, and my hope is that I will be able to attend and participate in a poster session. Until then, I will be spending part of the summer learning about extracting bee DNA and helping to test different extraction protocols. The other part of my summer will be spent learning about Yeast 2 Hybrid and associated techniques.

S2:13 Shout Out to Our STEM Mentors!

Poster Presentation-Arizona/Nevada Academy of Science Conference

Shout Out to Our STEM Mentors!
I think we all need to take a minute and thank our program mentors for helping us get to presentation-ready level with our research and creating the opportunity for us to attend symposiums and conferences. Over the past two weeks, I definitely understood the value in networking and communicating with mentors during the preparation stage for the conferences. I feel like the conference in Vegas went pretty well and I was able to take away what I need to work on in presenting to judges in the future. Aside from the conferences, I am working on making a connection with one of the ASU-West professors in hopes of being able to learn from her in lab. I'm very excited for this opportunity and definitely have Dr. Cotter and Dr. Chapman to thank for that!!!

S2:12 It’s GO TIME!

It’s Go Time!

What a week!!! I finished my poster Monday morning just in time to get it sent off to the printer. The rest of the week was spent getting ready for the ASU West Symposium and then the Arizona Nevada Academy of Science Conference. I am so glad that Dr. Cotter recommended going to the earlier poster session to get practice answering questions from the judges. It was definitely intimidating but great practice. I feel like the ASU West Symposium was great practice for the ANAS Conference. Next up is a five hour long drive to Nevada and a long night of squeezing in some homework, but it’s totally worth it!

S2:11 Seven Students, 2 Research Mentors,1 Van, and a Conference

https://aznvas.org/meeting/

Seven Students, 2 Research Mentors,1 Van, and a Conference
It's crunch time in the lab this week due to the upcoming ASU Symposium and the Arizona Nevada Academy of Science Conference. I just wrapped up my experiment and am almost finished analyzing my data. I am so glad that I was able to filter the rest of my samples after having the hiccup last week with the vacuum filter.  My poster is about ready to go, I just need to plug in my data and write the discussion section. I am definitely still nervous about having to talk about my project to total strangers because it usually involves maintaining eye contact and not zoning out in my head. 

S2:10 ....A Vacuum With a Sucking Problem?

....A Vacuum With a Sucking Problem?
I was really looking forward to running the rest of my samples this week but then I had an unexpected problem with the vacuum filtration system. Typically the system filters the water relatively quick (about 500mL under two minutes). For some odd reason, I noticed tons of air bubbles between the Buchner funnel and the porous plate. After two hours of trying to figure out what was wrong and testing a few things, we still haven't figured out what was wrong. What I was left wondering was if it is possible for the porous plate to be clogged. After talking with my current biology professor, he suggested soaking the plate in some type of acid. I am hoping to try this tip on Friday so I can finish filtering the water samples. In the meantime, I am trying to prepare my poster for the upcoming ASU Symposium. I am also in the process of registering for the AZ Nevada Academy of Science Conference. 

Source: https://chem.libretexts.org/Demos%2C_Techniques%2C_and_Experiments/Basic_Lab_Techniques/Vacuum_Filtration

S2:9 Just a Girl Living in a Plastic World

Just a Girl Living in a Plastic World
Going into this research project, I knew that people, including myself, use plastic in their daily lives. However, it wasn't until I started my experiment that I realized just how dependent we are on plastic. Think about it and ask yourself, "How many times today did you use plastic or plastic-related materials?" As for me, when I reflected on this, I realized I come into contact with plastic several times a day. Almost everyday, including today, my microwavable lunch comes in a plastic container. The fork I use to eat lunch is plastic. My water bottle is plastic. That's a lot of plastic!
Aside from pondering about how much plastic society depends on, I also began to start my project poster while I waited for the small glass petri dishes to get delivered by Amazon. Once they arrived, I washed them in distilled water and placed them into the laminar air flow hood to dry. It was a long process but pretty to look at.

S2:8 It's All About Control

It's All About Control
This week was all about brainstorming how to control for the independent variable since. Initially, distilled water seemed to be a good control but when I ran it through the filtration system and analyzed the sample, I saw a significant amount of microfibers and other unknown pieces of material under the dissection scope. As a result, I decided to try and filter a large quantity of distilled water several times in hope that I would see the quantity of microfibers and particles decrease after each filtration. After filtering the distilled water sample three times, I can still see the presence of microfibers and unknown pieces of material. I am going to try and filter the sample a few  more times and see if I can at least filter out all the microfibers.

S2:7 Out of the Lab and Off to D.C.

Out of the Lab and Off to D.C.
This past week was particularly hectic, but I would redo it in a heartbeat. I missed the first part of the internship week, because I had to fly to Washington, D.C. for a few days. I'm part of this political advocacy fellowship program where we are taught to advocate to our legislative members for the continuance of antipoverty programs like SNAP, MEDICAID, USAID, and the Global Fund. During the fellowship year, we fly out twice to Washington, D.C. and speak with Senate and Congressional Representatives. My favorite part of the policy summit was speaking with representatives on the importance of continuing to increase the funding for the Global Fund and USAID Fund, because Tuberculosis is the leading infectious disease killer among people around the world. Supporting programs like the Global Fund and USAID  can help other countries gain access to newer equipment and educational support to help with programs that reduce childbirth-related death rates, lower rates of TB infections, and many other programs. I also really enjoyed seeing how science can intersect with policy. After lobbying some AZ representatives, I flew back to Phoenix and picked up on the Microplastics project. Project highlights from this week included receiving the all glass jars and disseminating them out to fellow interns. I also spent some time brainstorming faster ways to collect water samples from around the Valley of Phoenix. I decided that I am going to ask my biology classmates to bring in samples. I think I will be able to collect water samples from all 13 villages in Phoenix. My goal is about 30 samples. We shall see what happens!

S2.6: I Saw Some Stuff Under the Microscope!!!!

 I Saw Some Stuff Under the Microscope!!!!

This week in internship, I started to run samples through the vacuum filter. So far, I have a total of seven samples (Shout out to everyone who collected and brought in a sample from their home!) and have tested four samples. The video above provides insight into the experimental process. Our attempt is to reduce contamination since theoretically, microfibers can be found in the air, and basically everywhere. With that being said, all the samples are ran in a laminar airflow. The first seven samples were collected in glass jars that contained black plastic caps, and this was due to lack of access to other collection containers.  Moving forward, we are now able to collect future samples in glass jars with metal lids to reduce water sample interaction with plastic. After running the four samples and analyzing them under the microscope, I saw what appeared to be microfibers in most of the water samples. I am looking forward to using non-plastic containers to collect and test more samples. 

S2.5: Off to an Exciting Start!!!

                                                                Off to an Exciting Start!!! 
Despite having three exams, this week went pretty well. I do have to give a shout out to my favorite study-buddy/tutor, Willie (see above video). In regards to the internship,I was super stoked when the filters arrived this week. Yesterday, I was able to run a sample test (referred to as "blank" of my experiment by using distilled water and filtering it through the vacuum filter for any microplastics and/or microfibers. Interestingly enough, after filtering and staining the filter, I saw the presence of microfibers when placed under the dissection microscope. The next time I am in the lab, I am going to stain a filter and place it under the dissection microscope without filtering any water. The purpose of running the blank and looking at an unused filter is to try and identify any potential sources of microplastic/microfiber contamination prior to filter the water samples. Basically, we want to be able to tell if the samples of water are being contaminated by our equipment during the filtering process. I am also looking forward to helping out with the Hermanas conference tomorrow! I really enjoy outreach!

S2.4: Would You Like Some Plastic With Your Water?

Vacuum filter that will separate the H20 and microplastics

Would You Like Some Plastic With Your Water?
This past week, I continued to work on my project background. I did some more research on existing studies regarding microplastics but still have only found one preliminary study on microplastics in global drinking water. I also continued to work with my project mentor, Matt, to collect the materials needed to conduct the experiment. The highlight of my week in the internship was Matt being able to find the same filter paper and pore size as outlined in the procedures we are following from another experiment. I should be able to start filtering the water by early next week.  So far I have four tap water samples collected from the central phoenix area. I am extremely grateful that two fellow stem-members agreed to bring in water samples from their kitchen sinks. My goal is to collect water from Northern, Southern, Eastern, and Western regions of Phoenix. We shall see what the next week in the lab brings!

S2.3: Snapshot of the Microplastics Research Proposal

(Reference 6)

The Presence of Microplastics in the City of Phoenix Public Drinking Water

Over the past decade several research experiments have indicated that society is now in the “Age of Plastic” and traces of plastic can be found virtually everywhere. Even when plastic seems to be broken down, not visible to the naked eye, it is still present on the microscopic level and polluting air, surface water, oceans, sediments, and marine life including types of seafood (Rochman et. al. 2015). Microplastics are also found in personal care products and machine-washed clothing (Wright and Kelly 2017). However, the question remains as to whether microplastics have polluted public drinking water. It appears as though only one public observational experiment surveying tap water has ever been performed. Research conducted by Kosuth et. al. (2017) collected 150 water samples from six regions on five continents, including parts of the US and found that 83% of tap water around the world contained microplastics, and of the samples taken in the United States, 94% of water contained microplastics. The researchers of the experiment expressed the need to further test geographical regions, and include more information about water sources and ground filtration methods prior to human use to better understand potential pathways of contamination. Phoenix is the 5th largest city in the country and the eighth fastest growing city in the United States (USA Today 2018). It is imperative to test Phoenix public drinking water for a number of reasons: Phoenix is one of the fastest growing cities; research on microplastics in tap water for the City of Phoenix is not publicly available and possibly has not be researched to date; and to contribute data to the emerging field of microplastics and its effect on human health.

Research questions

Are microscopic plastic particles and fibers present in public drinking water throughout the
City of Phoenix.

Are microscopic plastic particles and fibers present in filtered water and store-bought bottled water?

Hypothesis

Hypothesis 1: Samples of drinking water taken in the Phoenix Metropolitan Area will be positive
for microplastics.

Null Hypothesis: Samples of drinking water taken in the Phoenix Metropolitan Area will be
negative for microplastics.

Independent Variables: Location and type of  drinking water sample

Dependent Variable:  The amount of microplastics found in water sample

Control Variable: Vacuum Filter, Deionized Water (500mL), 2 mL Rose Bengal Dye,
Volume of Water Sample (500mL), water sample collection method, vacuum filter paper
(1.75 µm), laminar airflow cabinet, and Microscope.

Key Words:

Microplastics: Microplastics are small plastic pieces less than five millimeters long
which can be harmful (adapted from the NOAA)

Drinking Water: tap water which comes from public sources, residential homes, filtration
systems and bottled water

References

1.Comen, E. (2018, January 15). The fastest growing (and shrinking) states: A closer look. USA

Today. Retrieved February 08, 2018, from https://www.usatoday.com/story/money/economy/
2018/01/15/fastest-growing-and-shrinking-states-closer-look/1019429001/

2. Gerd Liebezeit & Elisabeth Liebezeit (2014) Synthetic particles as contaminants in German

beers, Food Additives & Contaminants: Part A, 31:9, 1574-1578, DOI: 10.1080/19440049.2014.945099

3.Kosuth, M., Wattenberg, E., Mason, S., Tyree, C., & Morrison, D. (2017, May 16). Synthetic

Polymer Contamination in Global Drinking Water (Rep.). Retrieved February 01, 2018, from Orb Media website:
https://orbmedia.org/stories/Invisibles_final_report

4.National Ocean Service. 2017. What are microplastics? National Oceanic and Atmospheric

Administration. United States Department of Commerce. https://oceanservice.noaa.gov/facts/microplastics.html

5.Rochman, Chelsea M., Tahir, Akbar, Williams,Susan L., Baxa Dolores V., Lam,Rosalyn,

Miller,Jeffrey T., Teh, Foo-Ching , Werorilangi,Shinta, Swee, Teh. Anthropogenic debris in seafood:
Plastic debris and fibers from textiles in fish and bivalves sold for human consumption. Sci Rep. 2015; 5: 14340.
Published online 2015 Sep 24. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585829/

6. http://www.phoenixasap.com/gfx/phoenix-villages.png

S2.2: Geeking Out Over My Research Project

Geeking Out Over My Research Project
This week I was able to gather more information on the first and only research project completed on identifying the prevalence of microplastics in drinking water. The research was conducted by Orb Media which is a professional nonprofit journalism organization that aims to provide data driven information to its stakeholders. For more information on Orb's background and mission see the link here: https://orbmedia.org/what-we-do
The research conducted by Orb Media stemmed from their question: "If microscopic plastic particles and fibers are found in the world's oceans and freshwater locations, could they be in the drinking water as well? The observational study collected 150 tapwater samples from cities and towns on five continents. The data revealed that microplastics were in fact found in drinking water but also called for more research to be done on the topic. The study was supervised by Sherri Mason, who is a prominent researcher in the field of microplastic pollution. Their study also provided the materials and methods for their research experiment. I was excited to come across this information, because my goal is to try and follow their method for consistency purposes. So far, I have gone through their report and noted the methods and materials section. My next step is to run a practice sample to familiarize myself with the methods and procedures. I will be collecting a tapwater sample from a residential home in Central Phoenix by the end of the week.
I am super stoked to find out how the data compares to the United States data. Based on the report, it appears that only samples from the West Coast, East Coast, and Midwest areas were taken but there is not mention of the Southwest or Arizona specifically. I believe it is imperative to have local data on microplastic pollution in drinking water. Phoenix is one of the fastest growing cities and in the top ten largest cities in the United States and therefore it is important to research microplastic pollution in tapwater sources.

References:
https://orbmedia.org/stories/Invisibles_final_report

S2.1: Back to the Lab!

Back to the Lab!
It's been really exciting to come start the spring semester, because I have been looking forward to the internship. My time back has gotten off to a rough start. I quickly learned the importance of para-filming sample plates for long-term storage. When I went into the refrigerator, I came to find that all my samples had been contaminated and dried-out. Luckily, there will be more creosote samples to tests, but this was definitely a hard lesson learned.
As for the rest of the semester, I have become interested in microplastics. I am hoping to work on a research project this semester which will allow me to test for micro-plastics in city drinking water. My interest in this topic came from reading a report on plastic contamination in the tap water of cities around the world. I am interested in finding out how data from the Phoenix area compares to national and global data.

S-STEM Intern Biography

Biography
My name is Samantha Faltermeier, and I am returning student working toward a second bachelor’s degree in Biology before I attend graduate school. My previous bachelor's degree is in Sociology. It is my third semester as a full-time student here at Phoenix College. My areas of interests are Biology, Public Health/Safety, and Public Policy. I have chosen this path for numerous reasons. I want to conduct research in a laboratory setting. I would like to use this research to help advocate for better health outcomes and health policy, because public policy affects the lives of people and research supports dissemination of unbiased information Lastly, this path will allow me to give back to my community by serving as a role model for future Hispanic women scientists.
In my free time, I enjoy hiking with friends and am a nationally certified EMT.