This document describes constructing deletion mutants of several genes in the uropathogenic E. coli strain UTI89. The key steps are: 1. PCR was used to generate gene disruption cassettes for spr, prc, eco293-sinI, and fimS. 2. The cassettes were electroporated into UTI89 cells expressing Red recombinase and plated on chloramphenicol to select mutants. 3. PCR confirmed successful disruption of the spr gene, replacing it with the cassette. 4. The cassette was then evicted by expressing FLP recombinase from pCP20, leaving only the gene deletion. 5. The process

1. To construct the deletion mutant of spr, prc, eco293-sinI and
fimS in uropathogenic E.coli (UPEC) strain UTI89
Adviser：Ching-Hao Teng Student：Yu-Chu Wu
Introduction Results And Discussion
Urinary tract infection is mainly caused by pathogenic bacteria, which can be treated by Preparation of PCR Gene Cassettes
antibiotics. As the overuse of antibiotics, increasing antibiotic resistance among pathogenic NK-spr PCR primers were mixed with the template plasmid, pKD3, and PCR
bacteria becomes a serious problem. Under this situation, it is necessary to new therapeutic components and then subjected to the PCR machine. After completion of the program,
methods other than the current antibiotic treatment for bacterial urinary tract infection. Therefore, the crude PCR product were mixed together and purified, then electroporated into UTI89
we want to explore the virulence factor which was caused by bacterial genome and figure out the by electroporation.
other ways to reduce or inhibit the function of virulence factor, then reduce the opportunity for Figure 2. shows an agarose gel of the spr-pKD3 PCR product prior to purification.
urinary tract infection. The product size appears to be approximately 1114 base pairs as expected. An agarose
Uropathogenic E.coli (UPEC) encodes numerous virulence factors, including various gel of the spr-pKD3 PCR product was show as Fig. 2.
adhesins and flagella. The ability of UPEC is invading or binding host epithelial cells to enhance
UPEC virulence and persistence within the urinary tract. UTI89 is an uropathogenic E.coli Fig. 2. An agarose gel of the spr-pKD3 PCR
(UPEC) and was isolated from a patient with an acute bladder infection. UPEC strain UTI89 can product
adjust to survive in the human body, especially in bladder. In the bladder, where they can cause First Well (on right side) contains 1 KB Ladder.
acute or recurrent urinary tract infection(UTI). The gene in the UTI89, which is including spr, Wells 1-2 contain 5 μl of PCR reaction mixture.
prc, eco293-sinI and fimS and their functions are different, such as fimS and eco293-sinI. The The product size of spr-pKD3 appears to be
function of fimS is binding to and invasion of human brain microvascular endothelial cells approximately 1114 base pairs as expected.
(HBMEC) by type 1 fimbria. The eco293-sinI is epidemiologically associated with urinary tract
infections.
Constructing of the spr, prc, eco293-sinI and fimS deletion mutants in uropathogenic
Escherichia coli (UPEC) strain UTI89 by a novel method of disrupting E.coli genes. Datsenko The Disruption of spr Gene
and Wanner (2000) recently described a method (using PCR products) for one-step inactivation Cells expressing the Red recombinase were electroporated with PCR gene
of chromosomal genes in E. coli. The method is a refinement of previous recombination-based cassette and incubated at 30°C and them were spread onto chloramfenicol plates to
methods of gene disruption PCR is used to generate a gene disruption cassette which directs the select for chloramfenicol resistant colonies. The colonies that continued to grow well
chloramfenicol resistance gene, flanked by FRT (FLP recognition target) sites, to a specific gene on chloramfenicol plates were tested to determine whether the desired gene disruption
in the E. coli chromosome by homologous recombination. A homologous recombination event had occurred. For checking the deletion mutant was successful or not, we done the
within the E. coli chromosome replacing the resident gene with the chloramfenicol resistance PCR. An agarose gel of the spr gene disruption in the UTI89 was show as Fig. 3.
gene is stimulated by the λ Red recombinase, expressed from the helper plasmid pKD46. The
Fig. 3. An agarose gel of the
chloramfenicol resistance gene is removed from the chromosome by recombination between its
spr gene disruption in the
flanking FRT sites, stimulated by expression of the FLP recombinase from the helper plasmid,
UTI89
pCP20. The process of deletion mutant was shown as Fig. 1.
First Well (on left side)
contains 1 KB Ladder and
Second Well contains 100 bp
Ladder. Positive control was the
wildtype UTI89, which size was
667 bp and negative control was
M.Q water. The product size of
spr gene disruption in the UTI89
( No.7,8,12,13,14,15,16,17,18,1
9,20,21,22,23,24) was 1172 bp.
Removal of the chloramfenicol Resistance Gene
Mutants were electroporated with pCP20 and transformants were selected on
ampicillin plates. All of the ampicillin resistant mutants, expressing the FLP
recombinase from the helper plasmid pCP20, were grown at 30°C. For checking removal
of the gene disruption cassette was successful or not, we did the PCR. An agarose gel of
the spr gene was disrupted in the UTI89, and then evicted the chloramfenicol resistance
gene from the strain UTI89 was show as Fig. 4.
Materials and Methods Fig. 4. An agarose gel of the spr gene was
disrupted in the UTI89, and then evicted the
An Overview of the Process of Gene Disruption：
chloramfenicol resistance gene from the strain
UTI89
First Well (on left side) contains 1 KB Ladder
and Second Well contains 100 bp Ladder.
Positive control was the wildtype UTI89, which
size was 667 bp and negative control was M.Q
water. The product size of evicting the
chloramfenicol resistance gene from the strain
UTI89 which was deleted the spr gene in the
UTI89 ( No. 1,3,8,9,14,17,23,25,30 ) was 242 bp.
Conclutions
During the summer pratical training, I
have learned many things and profited very
much from these experiences. I did many
interesting experiments that I have never
experienced (Table 2.). Otherwise, we tried
to detect the expression of protein which
was translated from transcription of prc,
ompA, fimA gene and dissect the mice for
their bladders and kidneys. I am lucky to
have the opportunity of coming to National
Cheng Kung University Institute of
Molecular Medicine for summer pratical
Table 2. The results of the summer
training and working with everybody in the
pratical training during the two months
laboratory.
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․Mulvey et al., 2001. Infect. Immun. 69:4572–4579. ․Datsenko and Wanner. June 6, 2000. PNAS. 6640–6645, vol. 97, no. 12.
․Foxman, B. 2002. Am. J. Med. 113(Suppl. 1A):5S–13S․Zhang et al., 1998. Nature Genetics 20:123-18.