Life as an undergrad in a research lab

I would like to start by thanking Dr. Eisen for giving me the opportunity to work in his lab.  This is my first time working in an “outside of class” lab setting and I feel that this project is perfect for undergrads looking for experience in a research lab.   The two mentors helping us, David and Jenna, are great teachers and are always anxious to share their knowledge with us inexperienced ones.  They taught us basic molecular biology techniques and procedures that are used in virtually every biology related lab around the world.

At first, the work was very interesting because I had no experience working in an actual research lab surrounded by actual scientists.  I was eager to soak up knowledge and asked plenty of questions.  When it came time for independent work and growing our own cultures I felt less motivated to do this because I excited to move on to the next stage of sequencing organisms.  As such, I decided not to work with any of the organisms I found and I am hoping to work with an organism that was grown and isolated by the group.

For my independent organism to work with, I am hoping to sequence Curtobacterium flaccumfaciens, a known plant pathogen that was tracked into the human built environment.  There aren’t any projects planned to sequence this organism or its relatives, so this seems like an ideal candidate for our project.  Its potential application to the built environment includes its ability to harm plants grown in the indoors, and/or harm crop plants that could make their way into our food.  This sounds like a very interesting bacterium to study because of the REAL world effects!

After we have sequenced the genome we will add it to a database, where other ACTUAL scientists can refer to the data we submitted and make their lives a little bit easier.

The Undergraduate Research Experience. . . so far

As my first REAL research experience, I had no idea what to expect.  Back in high school, I did a little research in my statistics class where I tested students’ memories based on the type of music they were listening.  Obviously, the data I collected wasn’t going to be research paper worthy.  Before I began working under the Eisen Lab, I thought to myself, “This is my chance to find some crazy new bacteria and name them after me!”  (Spoiler: None of us have yet found a novel bacteria.  Pretty disappointing, I know.)  Eventually, Winter Quarter came along and the undergrads finally began researching.

It was a pretty rough beginning for those first few weeks.  We didn’t know how to pipet – considering the only pipets we were exposed to were those plastic pipets in Chemistry labs; didn’t know the proper lab protocols for avoiding contamination; couldn’t perform a simple dilution streaking; didn’t know what was done and what needed to be done; and so on and so forth.  After finally coming up with a good system, the research went by smoothly.  And it actually has become a very fulfilling experience.  I’ve really come to appreciate the variety and unique characteristics of bacteria in the world.  Sometimes, I find myself looking at a surface and wondering what bacteria has made a home there.

Currently, each undergrad is isolating and extracting bacterial DNA from their choice of environment.  I took a couple of swabs from a restaurant’s chair, table, couch, and counter in Davis.  And I also took some swabs from a nursing home, and the Golden Gate Bridge in San Francisco.  I’m really close to submitting some of my 16S DNA in for sequencing.  I’m pretty excited to see what the results are.

Quick little slideshow summary of our work so far

I am a member of the Biological Undergraduate Scholars Program (BUSP), a science-enrichment program aimed at helping EOP students excel in their science courses and obtain research positions (thanks to them I obtained a position in Dr. Eisen’s lab, which had the added bonus of turning me into a blogger!).  I recently gave a presentation to my program coordinator and fellow second-year BUSPers that summarized the methods the Eisen undergraduates have used to isolate, purify, and analyze the 16S gene of various bacteria.  Dr. Eisen mentioned in my previous blog post that a summary of our work thus far would help our readers better understand our methodology and final goals, so I figured the presentation would be a good little placeholder until something more formal is written up

The Undergraduate Genomic Sequencing Project

I will admit that the slides are scarce on detailed information, and instead give very general descriptions. After all, I had to actually address the audience during my presentation and not just read off the slides!

Somebody ruined the party

Consider reading this first if you have no idea what we are doing

Last week I submitted 6 organisms to the UCD Sequencing Facility after several weeks of isolating and purifying their 16S genes. My initial submission of 5 organisms went somewhat smoothly, though something managed to sneak into two of my samples and mix the forward sequences I received (for each organism, we must submit enough for both a forward and a reverse reaction to be sequenced. We receive these sequences and align the complementary bases to generate a consensus sequence). I had really hoped this time would go much more smoothly than last, but unfortunately 4 of my 12 reactions, all forward reactions, were mixed and thus indecipherable.

I suspected the forward primer might have been contaminated at some point, and looking at my initial submission the only mixed reactions were forward reactions as well. Seems pretty clear to me that some little critter was able to find his way into our forward primer and rain on my alignment parade. I had hoped that I would have a larger list of organisms by this point, but here is what I have analyzed so far

  • Staphylococcus pasteuri
  • Enterobacter ?
  • Bacillus amylolequefaciens
  • Micrococcus luteus? (the most “interesting” organism I’ve found, based on GOLD results)

I brought in 5 new samples that I can hopefully begin culturing in the coming days, and in the near future I will resubmit the forward reactions that failed using a new batch of forward primer.

Color Changes in TTU3

I’d thought I’d take a minute to talk about one of the first microbes we isolated that has proven to be quite interesting. The microbacterium TTU3 which was isolated from a toilet biofilm first caught our attention as a brilliantly red colony in the middle of an otherwise rather dully colored plate. Although we know color is not necessarily an indication of environmental importance or any other quality other than color the color itself TTU (as it was called then) stuck in our minds and we paid attention as it went through the sequencing process.

While investigating the ideal growth temperature of TTU one of the things we noticed was that its color was temperature dependent. In a couple of side experiments, David Coil grew 5mL liquid cultures at 37ºC, room temperature and 4ºC and noticed that the colder the temperature was the brighter the red color and that at warmer temperatures the bacteria were whitish-yellow.

TTU’s color is temperature dependent but that’s not the whole story. This week, while growing overnight cultures to verify our stock culture, I noticed that after a dilution, the room temperature culture had turned from its usual pink color to the white-yellow characteristic of the 37º cultures but only at the edges of the biofilm that had collected at the bottom had changed color, the most dense collection of cells was still pink.

Upon further investigation I noticed the bright red plate (also stored at 4ºC) that we had made the overnight cultures from also had patches changed color in some places where it used to be red. What was most interesting about the white-yellow patches was where they were. On the streaked plate, the white appeared only at the beginning of the streak not in the single colonies at the end of the streak unless the colony had been picked for an overnight. In picked colonies, the white-yellow appeared only at the edges where the heat-sterilized wand had touched the colony.

These observations lead me to three conclusions: either the stock and plate have both been contaminated by a similar organism, the color change is also affected by density (perhaps quorum sensing in this organism is somehow tied to environmental temperature) or the color change is oxygen dependent (since we always limit the oxygen exposure of our 4ºC cultures to limit their growth while in storage).

I’m in the process of sequencing the 16s PCR product of these cultures so I will know soon whether the color change is due to contamination or not. If it is not contamination, figuring out the mechanism and conditions under which TTU3 will undergo a color change, may be an interesting side project to work on.

Bacterial Candidates: A Closer Look at the Contestants

After nearly ten weeks of learning our way around the lab, collecting samples, isolating organisms and sequencing their 16s ribosomal genes we are finally at the point where we are ready to choose our first candidate organisms for whole-genome sequencing!

The Plan:

  1. Choose candidate organisms
  2. Prepare a DNA library for each organism for Illumina sequencing
  3. Sequence and analyze genomes

Although we have a couple of pilot samples for which we are already preparing libraries, most of our organisms need to be screened for admittance into the elite group of “good candidate organisms.”

So who are these potential candidates?

Where were they found?

And perhaps most relevant to our project, have they been sequenced before?

Continue reading “Bacterial Candidates: A Closer Look at the Contestants”

Sequencing Reference Genomes

Sequencing reference genomes for various built environments requires a series of steps. The first part of the procedure is to sample an environment that possibly has interesting microbes. For an example one might sample a toilet, public tables, floors or human associated items. There are 2 ways to sample something, direct plating or indirect plating. Direct plating is done by directly swabbing the area of interest with a sterile swab then directly swabbing onto a plate. Indirect plating is done by swabbing the area then putting the swab directly into media and allowing growth to occur, then plating.

The next step after growing the samples is to dilution streak. Dilution streaking is a method used to isolate a single organism by systematically reducing the amount of bacteria in different parts of the nutrient plate. To dilution streak, first sterilize an inoculation loop and run it through a colony in the sample plate. Take the inoculation loop and create a heavy “pool” of microbes in one corner of the new plate. After doing this, sterilize the inoculation loop again and drag it through the pool creating a zig zag pattern. Repeat this step one more time using the previous pattern as the “pool”. This allows individual colonies to form.

After the individual colonies have grown, it is time to make over night cultures. To do this take 5ml of the appropriate media and with the sterilized inoculation loop, scrap a colony and put it into the liquid. After the overnight culture as grown we can extract genomic DNA by doing genomic preparations. Genomic preparation involves lysing the membrane and nuclei, which allows the DNA to flow into the solution. Cellular proteins and other impurities are then removed using salt-precipitating solution and isopropanol. Lastly, the genomic DNA is suspended in rehydration solution for further analysis. After this, we must confirm there is actually genomic DNA. To do this, we use an agarose gel. A mold of agarose solution is created with wells. The mold is placed in a “gel box” with TAE buffer. The genomic DNA and a UV-fluorescing dye are loaded into the wells and a current of 120 V is run through it for 20 minutes. This separates the DNA fragments. We then analyze the DNA fragments under a UV light. If DNA fragments are present, that means there is genomic DNA.

The next step is to do a 16S PCR. A PCR is involves ribosomal RNA which is highly conserved, and is done to amplify this ribosomal RNA. This procedure involves a master mix made up of Taq Buffer, Q Buffer, dNTP, P1 P2 Primers, Taq polymerase, water and DNA. Then we must confirm the PCR using agarose gel electrophoresis. Always have a positive and negative control and ladders. The gel allows us to see if the PCR was correctly done with a presence of a band at 1300bp and by looking at the size of the bands.

After 16S PCR is confirmed the PCR is cleaned up. This step requires Buffer NT-Bind 16S DNA to membrane, NT3 buffer-wash PCR reagents, dry silica membrane, and Elution- Buffer NE. The cleaned PCR is then TOPO cloned. To TOPO clone, use the Invitrogen protocol and kit. Take a few micro liters of PCR product and mix with TOPO vector, the enzyme ligates the PCR product to the vector. Then the plasmid is transformed into E.Coli by mixing the TOPO reaction with E.Coli and incubating on ice. The cells are then heat shocked to allows plasmids to enter. Then the cells are allowed to repair so ampicillian resistance can be expressed. Spread the transformation onto LB carbenicillian plates and incubate overnight. Plasmid preps are then made by first picking the correct colony and grow in an over night culture. Then the plasmids are isolated from the E.Coli following the Qiagen kit protocol. Various buffers are added to break down cell walls and precipitate protein then transfer into QIAprep spin column to capture plasmid DNA. Add more buffers to isolate plasmid DNA from genomic DNA then elute plasmid DNA into clean microcentrifuge tube by washing out the spin column.

The plasmid is then quantified using plasmid quantification procedures. Use a spectrophotometer to measure how much DNA was isolated during the plasmid prep. This shows us the amount of DNA that is in the sample, this is important because a certain amount of DNA is needed before the sample can be sent to be sequenced.  Use the dsDNA function and mix 1 micro liter of sample with 99 micro liters of water. Multiply the reading by 100 to take into account the dilution factor. After getting the readings the plasmids and/or the PCR is diluted to specific concentrations and transferred to the UC Davis DNA sequence center. A few days later the results are sent back to us. Using the Geneious program we edit and analyze the gene sequence. We copy and past the cleaned up sequence onto Blast website to compare the sequence of our 16s to other organism. Lastly we use Genome online database (GOLD) to see genome sequencing projects relating to our organism. We check the complete, incomplete and targeted projects to see what has been done thus far. From here we decide whether or not to go further into research.

Everyone choose a genome

Not enough reference genomes from the built environment?
Looking for ways to increase undergraduate participation in research?

The marriage of these two concepts seems fairly straightforward.   Bring undergraduates into the lab, have them culture microbes from the built environment, then sequence and assemble genomes… one per student.

That’s the process currently underway in our lab, with support from microBEnet and the Sloan Foundation.

In addition to the obvious goal of sequencing reference genomes we hope to develop a workflow and protocols for undergraduate isolation and sequencing of reference genomes.  Ideally this would be transferable to other labs, even classrooms.

Currently there are 6 undergraduates involved in this project, having commenced in January.   Since then they have learned sterile technique, basic microbiology/molecular biology protocols, 16S PCR, basic cloning, and how to analyze 16S rRNA sequences.  Each one has now processed their own environmental samples and we’re in the process of screening candidates for sequencing and starting to prepare Illumina libraries.

In addition to the science, this project has a strong outreach component.   The students will be blogging about their experience in this space and footage is being taken for a short “documentary” of the whole process.  Check back here for updates.