Grant Proposal
Undergraduate Research Project
Project Title:Identification of antibiotic resistant bacteria using 16S ribosomal DNA sequence analysis
Total Amount Requested: $704.10
Student Name:Julie Rengel
Student Signature:
Faculty Mentor Name: Dr. James Champine
Faculty Mentor Signature:
Date:September 2, 2003
Resubmitted:September 18, 2003
Antibiotic resistance in bacteria has become a major problem in clinical settings and in agriculture as well(Livermore 1995; Jank and Rath 2002). When antibiotics are given to livestock, the threat of antibiotic resistance in bacteria associated with the livestock is increased. The cost and threat to human life caused by these antibiotic resistant bacteria are immense(Gorbach 2001). A bacterium, FDM13, was isolated from a meat packing plant sewage lagoon by Josh Woloszynek that exhibits antibiotic resistance to penicillins, such as ampicillin(Woloszynek 2000). In addition, 16 non-enteric, ampicillin resistant isolates were obtained from a cattle farm by Kimberleigh Foster. They all produce an enzyme known as β-lactamase, which can inactivate the antibiotic(Bush et al. 1995). There are at least four classes of β-lactamase enzymes. Over the course of the summer while working in Dr. Champine’s lab, I have extracted both plasmids and chromosomal DNA from FDM13. I attempted to hybridize the plasmid and chromosomal DNA with probes for two types of β-lactamase genes using a Southern blot in order to determine where the gene is located. Knowing whether the gene is located in the chromosome or in plasmids will aid in determining if FDM13 has natural antibiotic resistance or if it was transferred from another organism. Kimberleigh Wilson is trying the same approach with her isolates. To compliment the hybridization approaches already employed, I would like to identify several bacteria. The goal of this proposal is to determine the identity of FDM13 and up to five of the cattle farm bacteria by 16S ribosomal DNA sequencing. This may be of value in understanding resistance in these organisms by comparison to closely related species.
16S Ribosomal DNA Sequence Analysis. In this method, the 16S ribosomal gene is amplified by PCR. The PCR products will be ligated into a vector, pCR 2.1. A TA Cloning Kit will be used to transform One Shot INVαF’ Chemically Competent E. coli with the pCR 2.1 vector containing the PCR insert. The transformants will be plated on LB media enriched with X-Gal and Ampicillin; the colonies that do not exhibit β-galactosidase activity (they will appear white) have been successfully transformed. I will extract the plasmids using a QIAprep Spin Miniprep Kit and send the PCR insert to Cornell University for sequencing. The sequence will then be compared to other organisms’ 16S sequences from NCBI Blast searches.
Budget
ItemDescriptionCost
TA Cloning KitTransforms E. coli with $298
vector containing PCR insert
(20 reactions)
X-GalIdentifies colonies with $43.10
β-galactosidase activity
QIAprep Spin Miniprep KitUsed to extract plasmid DNA$63
DNA sequencing$50/clone$300
Total$704.10
Budget Justification
The TA Cloning Kit with pCR 2.1 vectors and One Shot INVαF’ Chemically Competent E. coli are needed to efficiently transform the cells with PCR fragments. This particular cloning kit does not require extra reagents to determine if the fragments were successfully inserted into the vector, which is less costly. X-Gal is used to determine if the PCR fragment has been successfully inserted into the vector. If the transformation is successful, there will be no β-galactosidase activity and the colonies will appear white with the X-gal present. The QIAprep Spin Miniprep Kit is used to extract the plasmids from the transformed E. coli so they can be sent away for sequencing. The majority of the cost lies in sequencing the 1500 base pairs of DNA, for which approximately $50 per clone including shipping costs is budgeted. The 16S ribosomal DNA will be sequenced using universal primers that flank the gene on both sides. This will yield 600-800 bases of sequence that run in opposite directions. Both of these sequences will cost approximately $15 and an additional $15 is anticipated to repeat any questionable sequences. We would rather be conservative in cost estimates, than not have enough funds. Dr. Champine’s lab will supply media, ampicillin, tubes and reagents for PCR, and various supplies for electrophoresis.
REFERENCES
Bush K, Jacoby GA, Medeiros AA (1995) A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 39:1211-1233
Gorbach S (2001) Antimicrobial use in animal feed - Time to stop. New England Journal of Medicine 345:1202-1203
Jank B, Rath J (2002) Antibiotic-resistance management on the farm. Trends Microbiol 10:11-12
Livermore DM (1995) beta-Lactamases in laboratory and clinical resistance. Clin Microbiol Rev 8:557-584
Woloszynek J, J. Champine, J. Hoffrage, J. Miller, and M. Bluma (2000) Ampicillin-Resistant Bacteria Isolated From a Meat Packing Plant Sewage Lagoon. SAAS Bulletin 13:9-15