Identification of Enterococcus Species from 3 San Diego Beach Sand

Identification of Enterococcus Species from 3 San Diego Beach Sand

10/24/2016 Example good report – Fall 2016 1/5
Example good report – Fall 2016
Arun Sethuraman
Page 1
Lab 2 Mendelian Ineritance
Part 1
Overall Total: 15/15 Good job!
Total: 2/2 This is a good purpose statement. Try to make it a little more concise next time. For e.g. “The purpose of this experiment is
to understand the genotype-phenotype ratios that will be expected from a Mendelian dihybrid cross, as simulated using S. cerevisiae as a model system.” would be
easier and more appropriate.
Purpose Statement
The purpose of this lab is to be able to cross different strains of yeast, and to be able to observe a dihybrid cross and the different phenotypes that are
produced by these crosses, and their frequencies. The traits will be seen in color or growth with or without tryptophan in the media.
Total: 3/3 This is a very good introduction ­ very detailed and well composed. Good job! This covers all bases ­
introduction to Mendelian genetics, yeast, and the experiment.
Genetics is the study of how different traits are inherited and passed on from parents to their offspring. The concepts of genetics is based on the work
of Gregor Medel. Mendel suggested that all organisms have a pair of “particles” for each trait that is inherited, one from each parent. This, along side
of the work done by other scientists, has led to the acceptance of the chromosomal DNA being passed on to offspring.
There are two or more forms of traits; there are alternative forms of a single gene, which are called alleles. Each organism has two alleles for each trait,
one from the maternal side and the other from the paternal side. If they are the same alleles they offspring is homozygous but if they are different then
the offspring it heterozygous. Mendel looked at these crosses by only one trait at a time, he found that if he crossed pure breeding purple flowered
plants with pure breeding white flowered plants all of the F1 plants had purple flowers. Then with the F1 was crossed with itself to produce the F2
generation both purple and white flowered plants were produced. This meant that somehow the alleles for white flowered plants had to have been
present in the F1 generation but it was masked. From this Mendel concluded that one of the alleles of a gene is dominant in comparison to the other,
where when it is a heterozygote it is expressed over the other trait. The allele that is not expressed is known as the recessive allele.
10/24/2016 Example good report – Fall 2016 2/5
How an organism appears is known as its phenotype but a set of alleles is known as the genotype. The phenotype will be what ever the dominant allele
is unless it is homozygous for the recessive allele. The most common phenotype of an organism is known as the wild type, the less common variations
of that are given particular trait names. These variations are usually recessive to the wild type but in rare cases can be dominant to it.
In this lab we will be observing this using yeast. Yeast are simple fungi and refers to the unicellular phase of the life cycles of many different fungi.
The most common organisms called yeast are strains of a species called Saccharomyces cerevisiae. As fungi, they are classified as ascomycetes, The
Saccharomyces is the most commonly studied eukaryotic unicellular organism in genetics. This is due to them being easily grown in culture and it
reproduces both sexually and asexually. Asexual reproduction occurs mitotically where a progeny cell buds off the parental cell. Under harsh or
nutrient limiting conditions the Saccharomyces will go through sexual reproduction where it utilizes diploid cells and meiosis. These different mating
types are known as ‘a’ and ‘α’ (alpha). The gametes in yeast are known as shmoos, they are different from normal vegetative haploid cells only when a
cell of the opposite mating type is present. When the opposite mating type is present shmoos fuse, then they fuse their nuclei. This diploid cell can then
produce more diploid cells by budding. Eventually, a diploid cell will become an ascus and enter meiosis, this then produces four haploid nuclei that re
surrounded by thick protective coats and become spores, these spores are released and become new haploid cells. In this lab we will observe the
phenotypic frequencies and the dominant recessive traits through how the yeast grows when crossed on two different media.
Total: 2/2
1. I taped the copy of the table onto the bottom of the two petri dishes, the COMP dish has tryptophan and the MIN does not.
2. On the COMP plate I made a mixture for each of the type a strains with the type α strains. This was done by using the flat end of a clean toothpick to
transfer a small amount of the a1 strain of freshly grown cells onto the agar above the boxes on the template taped onto the bottom of the petri dish. I
repeated this for a2, a3, and a4 using a new toothpick for each of the strains. By doing this same procedure I transfered the α strain to each of the boxes
to the correct corresponding a strain and made sure they were mixed together.
3. I repeated this same procedure for the MIN plate.
4. I then drew a diagram of each of my plates and predicted the outcome of each of the squares.
5. Then I inverted both of my plates and placed them in the incubator at room temperature.
6. After the lab was over I washed my hands prior to leaving the laboratory.
Total: 5/5
For your notebook­Predictions
1. Before you leave, you should draw a diagram of each of the plates in your notebook and predict the outcome of each square for both plates
including both the genotypes and phenotypes. Remember, depending on what plate you are growing the yeast on, the phenotypes will be different!
Genotypic predictions for the yeast crosses.
10/24/2016 Example good report – Fall 2016 3/5
Phenotypic Predictions for the yeast crosses.
Haploid Diploid Grown on COMP Media Grown on MIN Media
RT R/R T/T Growth, Cream Growth, Cream
R/R T/t Growth, Cream Growth, Cream
R/r T/T Growth, Cream Growth, Cream
R/r T/t Growth, Red Growth, Cream
Rt R/R t/t Growth, Cream No Growth, Cream
R/r t/t Growth, Cream No Growth, Cream
rT r/r T/T Growth, Red Growth, Red
r/r T/t Growth, Red Growth, Red
rt r/r t/t Growth, Red No Growth, Red
For your notebook-Results
10/24/2016 Example good report – Fall 2016 4/5
1. Prepare a score sheet and record the color and growth phenotypes of each parent haploid strain and F2 diploid in each square.
Haploid Diploid Grown on COMP Media Grown on MIN Media
RT R/R T/T Growth, Cream No Growth, Cream
R/R T/t Growth, Cream No Growth, Cream
R/r T/T Growth, Cream No Growth, Red
R/r T/t Growth, Cream No Growth, Cream
Rt R/R t/t Growth, Cream No Growth, Cream
R/r t/t Growth, Cream No Growth, Cream
rT r/r T/T Growth, Red No Growth, Red
r/r T/t Growth, Red No Growth, Red
rt r/r t/t Growth, Red No Growth, Red
2. Which color phenotype is dominant?
Cream color is dominant.
3. Discuss if your previous predictions were correct. Why or why not?
10/24/2016 Example good report – Fall 2016 5/5
Most of my predictions were correct, except I thought I would have seen a little more growth on the MIN plate because I assumed the
tryptophan independent was dominant, but I observed that none of them grew without tryptophan, showing me that it was the other way around and the
tryptophan dependent was dominant.
4. How many different genotypes were there? How many different phenotypes? Do you have a 9:3:3:1 ratio? (No, you don’t have to do a chisquare test!)
There were about 9 different genotypes and only 4 phenotypes. Yes, you have a 9:3;3:1 ration when it comes to the genotypes.
5. What genetic principle is demonstrated in this experiment? Explain.
The genetic principle that is demonstrated in this experiment is the principle of independent assortment. This is because the cells color did not
depend on their tryptophan dependence and vice versa. This is shown when none of the crosses grew in the MIN plate, it did not matter if it was
red in color or cream.
Total: 3/3 Good job!
In conclusion we found that the yeast was shown to be following Mendel’s priciple of Independent assortment, but when we observed the outcome of
the crosses it also showed the principle of dominance when it came to the color of the yeast, but the yeast color did not influence its tryptophan
dependence. The only part of my hypothesis that was correct was that the cream color would be dominant over the red. I believe that some of the flaws
of this lab is it could easily become incorrect because of human error when making the crosses, as well as easily becoming contaminated if the top was
left off for an extended period of time. Based on the results of the experiment it shows that you have to consider different types of influences when
trying to predict how a trait will be inheirited and expressed.

Identification of Enterococcus Species from Beach Sand Samples

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Enterococci are important members of commensal microbial digestive communities in many animals and opportunistic pathogens that cause millions of infections annually. Their abundance in human and animal feces, the ease with which they are cultured, and their correlation with human health outcomes in fresh and marine waters have led to their widespread use as tools for assessing recreational water quality worldwide. The enterococci are most frequently used as fecal indicator bacteria (FIB), or general indicators of fecal contamination (Byappanahalli et al., 2012).


Global estimates indicate that each year more than 120 million cases of gastrointestinal disease and 50 million cases of severe respiratory diseases are caused by swimming and bathing in wastewater-polluted coastal waters. Swimming-related illness is attributed predominantly to exposure to microbial pathogens, which enter the water through sources, such as sewage outfalls, storm water runoff, animal fecal inputs, and human bather shedding (Abdelzaher et al., 2010). The Beaches Environmental Assessment and Coastal Health Act of 2000, 2012 (BEACH Act) requires all coastal states and territories to monitor the water quality of its recreational coastal beaches for pathogens that are harmful to human health using guidelines established by the U.S. Environmental Protection Agency (U.S. EPA). This includes monitoring for the presence of Enterococcus bacteria (ENT) including the species faecalis and faecium. Although harmless strains of the bacteria are present in the human digestive system, more virulent and antibiotic-resistant strains also exist that when ingested in high enough concentrations can cause significant gastrointestinal distress and potentially other illnesses including skin-related symptoms such as rash and itching. Further, the presence of ENT is also indicative of the presence of other fecal bacterial pollution. As such, ENT results are referenced as fecal indicator bacteria (FIB) used to assess the possible presence of other such bacteria (Coakley et al., 2016).


Fecal indicator bacterial concentrations in beach sand are not routinely measured despite the possibility that beach sand may act as an important reservoir for microbial contaminants. If fecal indicators are being concentrated in beach sand, then fecal-born pathogens may also be accumulating at this site therefore raising the question of whether contact with sand poses additional health risks related to beach use. (Bonilla et al., 20017). Recent studies have shown that ENT is widespread in beach sands along the coast of California, Hawaii, North Carolina, and Florida (Coakley et al., 2016). As such, the concern is that because current FIB guidelines do not monitor the presence or concentrations of Enterococcus (or other pathogens) in beach sands, sand contamination may serve as another human exposure route that can cause illness and be contaminated in the same manner as coastal waters, i.e., sewage outfalls and runoff (Goodwin et al., 2012)


One of the primary methods used to detect the presence of Enterococcus or other FIB is through time- consuming culture methods. In addition to time, misidentification of ENT sub-species faecalis and faecium is common with this culture technique. As a result, polymerase chain reaction (PCR)-based methods are being employed that more quickly identify and quantify bacterial species present in environmental samples. One PCR based genotyping technique that provides rapid identification of ENT sub-species is PCR-Restriction- Fragment –Length Polymorphism (PCR-RFLP). Genotyping is any method that allows researchers to look at a particular location in a genome and be able to discriminate either individuals from each other or a reference sequence and/or determine their allelic composition.


The PCR-RFLP method is based on the analysis of particular genome regions which are first amplified and then   subjected to restriction analysis. The method thus consists of two steps; PCR amplification, followed by enzymatic digestion or restriction. The genome region to be analyzed is selected in line with the purpose of the particular analysis. It can be a species-specific sequence, which may allow for simultaneous genus and species identification, or a region-enabling phylogenetic analysis. The region selected as a molecular target should include a variable sequence, which enables the differentiation of the analyzed strains. Primer sequences are designed at conserved sequences flanking the variable region. The restriction enzymes for subsequent digestion reaction usually comprise a frequent-cutting enzyme which recognizes 4-nucleotide sequences. The appropriate enzyme is selected on the basis of the analyzed sequence, or experimentally if the sequence of the product is unknown. The advantage of the PCR-RFLP method is that it enables simultaneous species identification and strain genotyping. The method is widely utilized in studies on account of its high reproducibility, straight forward analysis and ease of implementation. Below is a figure demonstrating the use of PCR-RFLP to distinguish two variants of the -360CG region in the gene encoding deoxycytidine kinase of humans.



Purpose and goals of this lab:

In this lab, you are being provided with DNA samples obtained from sand at three San Diego beach locations. See the map below. All sand samples were collected from the intertidal zone area (area that is above water at low tide and under water at high tide) at each location. Four hundred grams of sand was collected from each site and all DNA was extracted using a FastDNA Spin kit for soil and a FastPrep instrument (MP Biomedicals, Santa Ana, CA) according to the manufacturer’s protocols.


Your goal is to use the PCR-RFLP procedure with gene cloning to determine if ENT species are present and if so, are their sub-species within the samples. You are also interested in determining whether there are differential spatial profiles of ENT bacteria at the sampling sites. In particular, you want to determine if the proximity to the storm drain outfall is a source of sand bacterial contamination.


Design the experiment:

It will be up to you (as a class) to design an experiment that will help you determine the goals stated above. Below is the list of items you have available to you.


  • DNA samples from sand at the three sampling locations


  • PCR-RFLP protocol that has been used in previous experiments that targets the 16S gene of ENT species


  • PCR reagents—Forward and Reverse primers, DNA polymerase, buffer, dNTPs, MgCl2, and thermal cycling machine.


  • Restriction enzymes MlyI for identification of species


  • Agarose gel powder, buffer, and gel apparatus with power supply to observe presence and size of a) uncut PCR products b) MlyI cut PCR products. Size control is 100 bp ladder




PCR Protocol-

The 16S rRNA gene is to be amplified by PCR with the following primers: forward primer U968 [14], 5’- AACGCGAAGAACCTTAC -3’; reverse primer L1401 [14], 5’- CGGTGTGTACAAGACC C -3’.


PCR amplification is carried out in 20 μl volumes containing 1 μl of potentially contaminated DNA from three sand samples and 19 μl of amplification mastermix.


The mastermix contains the following components (concentration): 10 x PCR buffer, dH2O, 0.2 mM (each) dNTPs, (Fermentas), 25 mM MgCl2 (Fermentas), 10 pmol each primer and 1.25 U of Taq DNA polymerase (Fermentas).


  1. Prepare the mastermix in a 1.5 ml Eppendorf tube using the 4.1x portion of the table. Pipette very carefully.


Component 1X 4.1X
dH2O 11.5 µl 47.2 µl
10x buffer 2 µl 8.2 µl
dNTPs 2 µl 8.2 µl
MgSO4 1.5 µl 6.1 µl
Reverse Primer 1 µl 4.1 µl
Forward Primer 1 µl 4.1 µl
Total 19 µl 77.9 µl


  1. Add your completed mastermix to the tube labeled Taq DNA Pol (amount of Taq is too small to accurately pipette). Mix gently by pipetting.
  2. Label 4 PCR tubes for the 3 samples and 1 control.
  3. Add 19 µl of the mastermix to each tube. Again pipette very carefully to avoid running out of mastermix.
  4. Add 1 µl of the appropriate DNA to each tube according to your numbering (Sample 1, 2, and 3). Add 1 µl of dH2O to the control tube.
  5. Bring your samples to the PCR machine at the front of the lab.


The PCR amplification is performed in a C1000 thermal cycler (Biorad) using the following program:

  • 95˚C for 3 min to denature the target DNA
  1. 95˚C for 10 s
  2. 58˚C for 20 s Repeat steps 2-4 for 30 cycles
  3. 72˚C for 30 s
  4. 72˚C for 5 min for a final extension
  5. 4˚C hold until testing.


An amplicon of 433 bp is expected if the samples are contaminated with any bacteria.




For Digested Samples:

  1. Spin down PCR tubes to collect sample at the bottom of the tubes.
  2. Label 3 eppendorf tubes for samples 1, 2, and 3.
  3. To each tube add the following:

RFLP Protocol- 20 ul reaction

  1. 10ul PCR product
  2. 2ul 10x CutSmart Buffer
  3. 5 ul H2O
  4. 5ul MlyI restriction enzyme
  1. Incubate at 37˚C for 30 minutes
  2. Incubate at 50˚C for 5 minutes to inactivate MlyI
  3. Add 4 µl 6x SYBR Green Loading Dye
  4. Spin down to collect sample at the bottom of the tube


For undigested Samples:

  1. To the remaining PCR sample (Samples 1, 2, and 3) add 2 µl 6x SYBR Green Loading Dye
  2. Add 4 µl 6x SYBR Green Loading Dye to the control tube.


Gel Electrophoresis:

  1. Prepare 50 ml of 3% agarose in sodium borate as in previous lab.
  2. Load your samples onto the gel and run as in the previous lab.




8 µl of DNA ladder 10 µl Sample 1 PCR + 2 µl loading 10 µl Sample 2 PCR + 2 µl loading 10 µl Sample 3 PCR + 2 µl loading 20ul Sample 4 PCR (H2O control) + 4 µl loading 24 µl of MlyI cut Sample 1 PCR product 24 µl of MlyI cut Sample 2 PCR product 24 µl of MlyI cut Sample 3 PCR product