Signature-tagged mutagenesis (STM)

The term "signature-tagged mutagenesis" (STM) refers to a technique that allows for the simultaneous screening of numerous different mutants. The method is accomplished by applying a special DNA sequence known as a signature tag to each of the implicated bacteria. Initially, this method was referred to as TN mutagenesis screening and it was used to find Salmonella bacterial genes crucial for survival (Hensel et al., 1995, p. 400). The method was developed to use 40 base pairs per DNA sequence. There were two main steps to the entire process. The first entails generation of a pool of transposon molecules that contains the specific sequence used to produce a tagged mutant library. When the method was established made use a complex mixture of the two strands of DNA tags which carried the transposon and mini TN5Km2 was used in sequencing the library. It contained 20 base pairs on both ends. The double stranded DNA was then incorporated into this plasmid through ligation as well as maintenance of E.coli as the pools of transposon were tagged by random signature sequencing (Hensel et al., 1995, p. 400). These would then be transferred into a specific as well as suitable host through the process of conjugation to produce many mutants. The second phase encompasses in vivo screening of the generated library. Here, the tags were determined before PCR being used to amplify, label, as well as hybridize the blot DNA probes prepared from the mutant which yields a signal on autoradiogram. These were then assembled into a different 96 well pool and exposed to a process for selection which involves infection of an animal. After this stage, often the mutants were recovered and put in PCR for amplification to generate labeled probes that represent the tags. These were hybridized from the output as well as input pools to dot blots followed by identification of the tags host in the output which in return enable isolation of the strain that did not survive the infection process (Hensel et al., 1995, p. 400). The sequence of the nucleotide from the recovered mutants is determined through sequencing method. It is also imperative to note that STM depends on mutant propagation capacity in vivo and the method requires several parameters to be considered to obtain reproducibility of the identified result. At the same time, there were several bacteria that in STM. Escherichia Coli (E. coli) is mostly used in various studies for identification of its virulence using signature-tagged mutagenesis. There were many organisms and body parts in which different strain of these bacteria colonies. While most of the strains were commensal, some researchers have focused on avian pathogenic E. Coli (APEC) which leads to the development of a disease (Nakazato et al., 2009, p. 483). The organism often enters and colonizes avian respiratory system resulting in localization of infections like pneumonia and arisacculitis. In some cases, APEC spread to other internal organs leading to the development of peritonitis, perihepatitis, and pericarditis and salpingitis diseases. There were several bacteria that were associated with virulence of APEC including the acquisition of iron, adhesins, and toxins. However, the mechanism that underlies its pathogenesis is still not well understood. As such, various researchers have focused their studies on identify the gene involved in this process. Different methods have been used, for example, fluorescent induction, transcribed sequencing and vivo expression technology (Ewers, Janßen, and Wieler, 2002, p. 381; Janßen et al., 2001, p. 373). Recently, scientists have been focusing on STM to identify all the genes that may be present in APEC genome. The result has been used to report the novel and known genes that were involved in the pathogenesis of APEC wild-type. As such, it will be imperative to focus on the process undertaken to identify these genes using STM.


Materials and Methods
Bacterial strain, plasmid, and conditions for growth
To carry out the study, researchers have often used E.coli strain IMT5155 for infection studies and STM analysis and mutant construction. The strain is known to cause coliseptisemia disease. It is isolated from an internal organ of four-month-old laying hen (Mellata et al., 2003, p. 538). Most of the avian used must have shown symptoms of coliseptisemia. Further, IMT5155 was used in phylogenetic analysis of one hundred as well as fifty APEC strains. It is known to carry some of the major virulent genes as demonstrated by PCR and hybridization experiments.
To carry out the procedure, a spontaneous a resistant mutant was generated using nalidixic acid by grown the above strain there in the presence of antibiotic as well as plating of 109 CFU. After this procedure had been performed, the next step involved confirming that the strain still has full pathogenicity using various chicken models (Johnson et al., p. 321). The bacteria were then cultured in 37 degree Celsius in LB broth or agar that contains suitable or important antibiotics at various concentrations. It is imperative to include controls in the process. Usually, other strains such as ITM5104 was isolated from feces of healthy egg laying avian and confirmation made to ensure that it did not harbor any of the previously obtained virulence related to APEC strains. This was used to serve as a negative control (Germon et al., p. 119).
Primer and manipulation of nucleic acid
PCR primers P3 and P5 were used to amplify the genomic DNA. Primer P2 and P4 were applied in making labeled DNA for probing the colony output and input pools. Others were used to carry out sequences as will be described in the discussion (Ronco et al., 2017, p. 13). The nucleic acid was manipulated according to the standard biological technique standard.

Tag Selection and development of Library of APEC mutant
Tagged plasmids were provided, and each had approximately 40 base pairs containing random sequences which were flanked by about 20 base-pairs invariant sequences that function as primers used to amplify tags of the E. coli strain is transformed with the specific plasmid which already contains signature tags, and several transformants were obtained. At the same time, further screening was carried out by hybridization method with cognate labeled tags. Later, experiments involving cross-hybridization were performed, clones which demonstrate strongest signal were collected, and further investigations performed to confirm the success of this procedure (Ronco et al., 2017, p. 13). Only those transformants with single labeled tags were taken through further confirmations. According to Badger, Wass, Weissman and Kim (2010), this was also be achieved by transforming a pool of random signature-tagged transposon in plasmids. This is followed by subculturing the colonies to obtain optical density and plasmids were mobilized through conjugation. Transconjungations were plated onto LB or agar plate containing casamino acid and thiamine, nicotinamide as well as glucose supplemented with kanamycin.
Finally, purification of plasmids carrying specific tags was performed from the clones. They were then transformed individually into donor strain E. coli. The process was allowed to take place to log phase. For the researchers to create transposon of the tagged mutants, an equal concentration of donor as well as recipient cells was mixed, and centrifugation carried out (Badger et al., 2010, p. 272). The supernatant is discarded and cell resuspended in a solution of magnesium sulfate. The next step involved placing them onto LB agar plate and temperature adjusted to 37 degrees Celsius for incubation for about eight hours. Colonies formed were collected, and resuspension is performed in phosphate buffer saline. This was followed by plating onto LB agar with an antibiotic such as nalidixic acid and kanamycin. The following stage entails overnight selective growth. All the identified single colonies were collected as well as resuspended in approximately 300µl LB supplement. An overnight incubation is allowed, the following morning, 15% glycerol is added to a suspension of colonies and mutant collected and stored at a temperature of -800C in microtiter plates.
Characterization and Assembly of the Pools
The procedure also involved identification of signature tags that hybridized sufficiently well to be used in the screening assay step where colony blots were carried out. The process entailed using positively charged nylon membrane which was replica-stamped microliter well from each pool of bacteria (Antão et al., 2009). The DNA from colonies from bacteria was then denatured through a process called alkaline lysis followed by neutralization and membranes washed extensively using standard saline citrate. These membranes were then incubated together with the labeled probes generated. These were obtained from homologous signature tag DNAs applying the stipulated standard procedures. However, some of the clones and probes failed to hybridize so that it was difficult to visualize them on the autoradiograph. This followed the determination of frequency as well as an approximate position of transposon insertion in E.coli. Further, genomic DNA was digested using HindIII from the clones; this was then used to carry out Southern blot probed with isotope 32 of phosphorous (Antão et al., 2009).
Animal model, STM screening for transposon mutant library and In vivo as well as in vitro competition assay
An animal model that used chicken was utilized to perform STM analysis for E. coli septicemia. Leghorn specific chickens free from pathogen were inoculated into a trachea with a suspension containing dilutions of IMT5155 strain for preliminary investigation. Preparation of inoculants is the next step that follows. The frozen mutants (those put in -80 degree Celsius) were taken and subcultured carried out through the transfer of ten microliters from every well a different 96 well plate with 300 microliters of nalidixic acid and kanamycin (Antão et al., 2009). After culturing overnight, each plate is pooled and ten milliliters of bacteria added to ninety milliliters of LB. This is incubated at 37 degrees Celsius and aeration enabled to optimize the density. The cultures were then diluted in PBS, and 0.5 milliliters was drawn from the mutant pool and used in the process of infecting the leghorn chickens which were later killed and isolation of the bacteria completed from the spleen. The organs were broken down in the sterile plate on PBS then agar was supplemented with nalidixic acid and kanamycin. After incubation overnight, 5-milliliter aliquots of the pool were placed in ten milliliters of PBS and allowed to form output pool (Antao et al., 2008, p. 363). This was then used to make genomic DNA and those mutants that show a reduced level of hybridization signal were used an individual to quantify their relative rate of growth.
Both intro and in vivo competition assays were done by first mixing the wild-type and mutant strain at an equal ratio. For the research to perform the in vitro investigation, the bacteria were incubated at 37 degree Celsius in LB broth for four hours and then plated to media A and B. The LB plate had kanamycin while the other did not. In vivo competition procedure was accomplished using four chickens which were infected with appropriate dilution, and after two days of infection, livers, heart, spleen, kidney and lungs were collected, weighted and homogenized (Antao et al., 2008, p. 363). The obtained dilutions were put on a plate with media A and B. This procedure is performed to select mutant and aggregate number of bacteria respectively. The next phase entails calculation of mutants to get competitive index by dividing the output (wild-type/mutant) ratio with input (mutant/wild type).

Figure 1.0: The diagram illustrates the general process of signature-tagged mutagenesis (source: (Antao et al., 2008, p. 363)
Sequencing Methods and Identification of Auxotrophy
Restriction enzymes were used to digest the genomic DNA obtained from desired. The enzymes used were incapable of cutting transposon insert (Antao et al., 2008, p. 363). Digoxigenin-labeled probes were used to probe the DNA after electrophoresis. This was produced from BamHI, a digest of pUTminiTn5Km2 followed by labeling with digoxigenin. The next step involved confirmation of proper insertion of DNA of interest fragment into the plasmid. Further, the procedure entailed purification of plasmid DNA, sequencing followed.As identified in the introduction, STM depends on the ability of the mutant to multiple (Antao et al., 2008, p. 363). As such, CAA medium was modified to determine replication ability of the clones.
Manipulation of DNA and Sequence Analysis
DNA obtained from the chromosomes of every mutant was digested. Again, specific restriction enzymes were used to accomplish this goal open the flanking for insertion of the transposon. These enzymes do not cut within the transposon. The fragments that arise from this process were ligated and allowed to undergo inverse PCR reaction with primers P7 and P9. DNA is manipulated, and transformation performed using standard method. Modifying agents, such as restriction enzymes were used (Dozois, Daigle, and Curtiss, 2003, p. 249). These were obtained through molecular biochemical. The next step is to do hybridization with digoxigenin-labeled probes. A considerable amount of nanogram of DNA is attained and used for hybridization experiments. Next, positively charged nylon membrane is used to perform blotting as recommended by the manufacturer (Dozois, Daigle, and Curtiss, 2003, p. 249).
Labeling and hybridization for identification of mutant disappewered from Recovered Pools
Tagged sequences were obtained by PCR method to reduce the background of hybridization. These sequences were utilized as the target DNA for dot blotting instead of using the whole plasmid with tagged transposon. Conserved sequences of tags were removed using gel electrophoresis technique. The following phase entails amplification of DNA tags. To achieve this objective, five nanograms of DNA or 5 microliters of genomic DNA obtained from the input and recovered pools use as a template in the initial PCR round taking into account all the important condition for to oversee successful completion of this process (Fuller, Kennedy and Lowery, 2000, p. 26). The amplified tags from recovered pool and inoculum were compwered through labeling then with digoxigenin-dUTP using PCR probe generation kit based on the instruction indicated by the manufacturer. Purification is the next stage and after the products from PCR were labeled 50 microliters mixed with 50 pmol of the primers. This was also attained by mixing with digoxigenin-dUTP and sufficient concentration of the stock solution of deoxynucleoside triphosphate for the first cycle of PCR (Fuller, Kennedy and Lowery, 2000, p. 26). The products were then digested with HindiIII, a restriction enzyme, to form enough volume and electrophoresis is performed. The region with identified to contain labeled tags was cut from the gel. They were used as probes for hybridization process. On the other hand, cognate from original plates with tags were used as templates to amplify the primers. Additionally, identical dot blotting procedure is prepwered through the transference of the amplicons to a positively charged nylon membrane (Fuller, Kennedy and Lowery, 2000, p. 26). After hybridization is performed, and mutants that demonstrate signals with the probe obtained from input but not recovered pool were chosen for more evaluation.
Transposon Insertion Sites identification followed by analyses of sequences
All the transposon insertion points were amplified through arbitrary PCR. The first round involved primers Arbi1 to Arbi5. These were used in combination with specific primer P9 specific to the transposon. In the subsequent step, one microliter of each product of PCR was used during the second PCR in which primers Arbi2 and P6 were used. Arbit2 is similar to 5′ sequences of the above arbitrary primers (Dziva et al., 2004, p. 631). Genomic DNA from wild type was used to function as the negative control of the experience. Further, products from the second PCR round were purified and sequenced. This was followed by the analysis of the data using the public protein and DNA databases such as BLASTIN and BLASTX (Kukavica-Ibrulj and Levesque, 2014, p. 542).
Analysis of component that causes virulence in the strain
The most considered components associated with virulence of APEC include the lipopolysaccharide (LPS) which was attained by digestion of proteinase K of the entire cell (Han, 2014, p. 489). The samples of separated LPS were obtained through gel electrophoresis using 15% sodium dodecyl; sulfate polyacrylamide according to the standard procedures.
Results and Discussion
Creation of STM transposon
This stage involved generation of a signature-tagged library of a transposon of APEC. From the strain IMT5155, a library of signature-tagged mutants is constructed using non-cross reactive tags that were first prescreened. These were then amplified as well as efficiently labeled as explained in the material and method section. Based on the information gathered from the literature 1% of the mutants have been identified to be resistant to ampicillin (Darwin and Miller, 2009, p. 53). This is an indication of the presence of suicide plasmid with transposon integrated into the chromosome or within the cytoplasm of the bacteria cell. Other mutants were found to have lost kanamycin spontaneously lost resistance to kanamycin and were not included in the process of constructing the library. Southern blotting was performed to determine the transposons that contained insertional hotspots using randomly selected mutants, digested and probed with fragment gene resistant to kanamycin. After hybridization, it the researchers noticed a different pattern from the mutants (Darwin and Miller, 2009, p. 53). In general, the result indicated a wide distribution of insertion of transposon sites in the mutants.
Screening APEC mutant Library using Chicken Model
In this step, preliminary trials were done aimed at obtaining optimal protocol to screen in vivo tempered with mutant obtained from STM. The important parameters considered included determination of the effective 50% infective dose selecting those pathogens that were a highly infectious strain, the most suitable duration for infection and identification of optimal time during which the bacteria can be reisolated from the various internal organs (Darwin and Miller, 2009, p. 53). Inoculation of 108 folds was used to show reproducibility of the disease and its progression allowing isolation of more than 85% of bacteria from the chickens. At the same time, the control strain, IMT5104 was not recovered regardless of the concentration used for the inoculation from the internal organ. This is because the chickens did not show any clinical symptoms of the diseases. The following phase involved random selection of pools that were used to infect the animals, and IMT5155 bacteria were re-isolated from organs such as spleen after 2 and three days following the time of infection (Mecsas, 2002, p. 36). In most cases, mutants were absent from the output pool as shown in figure 2.0. However, the researchers identified 19 mutants that were capable of causing infection and were used to perform competition assay.

Figure 2.0: The figure shows results of STM from the representative pool. It also illustrates growth of the mutant in vivo and in-vitro (Source: (Mecsas, 2002, p. 36)
Generation of mutations, Characterization, and identification of interrupted genes
Mutants were constructed by targeted gene disruption as follow. The process of cloning analysis of identified gene disruption. The internal open reading frame (ORF) is generated based on the sequence obtained through CPR mechanism. In this phase, insertion site for the plasmids for the selected mutants is identified through amplification of their flanking DNA regions (Mecsas, 2002, p. 36). This is achieved by depending on PCR that was arbitrarily primed. The amplified DNA products were then sequenced into a base pair in the range of one hundred and fifty and seven hundred and fifty base pairs. For the analysis of the sequence, BLASTN or BLASTX hits were recorded. The genetic locus with the closest match to interrupted sequence by the transposon was obtained from each mutant (Mecsas, 2002, p. 36).
Quantifying Virulence of the selected Mutant by in vivo Competition assay
This step was done to validate the findings of STM screening procedure as well as carry out quantification of the extent of virulence of attenuation of each mutant. These were chosen depending on their classification. The selected mutants were mixed with pwerental strain. An equal amount of each was used followed by inoculation into 4 distinct avian birds through intratracheal route. The total number of bacteria was re-isolated from spleen, heart, liver and lungs as well as kidney after 2 days (Mecsas, 2002, p. 36). This was followed by calculation of competition index as described in methods and material section above.
Sequencing the Genes Cluster in IMT5155and Identification of Unknown Genes
The genes associated with virulence of APEC were completely sequenced in IMT5155. The following was the genomic organization of the region: putative outer membrane, periplasmic chaperone, and adhesin genes (Ron, 2006, p. 29). Other genes identified were associated with the production of polysaccharides and iron intake in the host organization. Several mutants were determined to contain transposon insertions for putative or unknown functions. These were subjected to further testing using the in vivo competition assay. Some of these genes include yobB associated with the production of putative amidohydrolase. Another is ydeH which is also related to the generation of putative diguanylate cyclase domain involved in cell adhesives of the bacteria (Ron, 2006, p. 29). Certain locations of the strains were associated with genes such as EA9F4, EA2E10, and EA1A9 which had insertion genes whose function were previously unknown. These were used in breaking down the lactamase ring making conferring the bacterial resistance to antibiotics.
Determination of APEC Virulence
The researchers focus on the different mechanism associated with virulence of the E. coli. Synthesis of different exopolysaccharides is one of those. This phase also entails identification of insertion found in attenuated mutants focusing on the genes responsible for the development of extracellular polysaccharides. Further, the stage entails the determination of mutant with insertions in different loci (Rollins et al., 2005, p. 8). For experiments that use the APEC, the research identified two loci that encode protein need for translocation of E. coli that were located in the inner membrane. These were group II proteins, and in the previous study, K1 has been identified, group II antigen which provides further evidence for biosynthetic processes involved in APEC pathogenicity (Rollins et al., 2005, p. 8).
Other loci such as M03G02 have been demonstrated to harbor a disruption in a sequence associated with putative colonic acid. It has been realized that the locus is found attenuation in most experiments. The region is responsible for the formation of other biological acids such as D-glucuronic acid and pyruvate. These components were used by the bacteria to form mucoid matrix thick enough on the cell surface that aid in pathogenesis (Vidotto et al., 2007). As such, this adds to the advantages the bacteria have that contributes toward virulence. Further, it is imperative to note that this information provides evidence for of colonic acid and seriousness of pathogenicity.
Discussion
Factors Associated with Genes Identified that cause Virulence of APEC
Exopolysaccharides Synthesis
It was identified that STM mutants harbored a disrupted sequence associated with synthesis of putative colonic acid glycosyl transferase. The acid was attenuated which indicated that this process was important for the bacteria to survive in the different organs of the host (Vidotto et al., 2007). Besides these, E.coli strained IMT5155 was found to generate exopolysaccharides that contribute to adhesion of epithelial cells. As such, according to various researchers, thus suggested the ability of the bacteria to protect itself from host environmental stress, for example, body temperature as well as macrophages. Therefore, various studies have provided evidence of association of exopolysaccharides with virulence as well as the fitness of this pathogen into the infected chickens (Stordeur et al., 2002, p. 236).
Lipopolysaccharide Synthesis which contributes to Viability of APEC
Genes associated with the production of lipopolysaccharides were part of the primary entities identified which form the bacteria outer membrane (Han, 2014, p. 489). The molecules were found to be made of oligosaccharide and polysaccharides specific to antigen O. Different sources indicate that research has realized some of the mutants isolated from STM contain transpositional insertions in genes that carry out the synthesis of LPS (Stordeur et al., 2002, p. 236). The mutant tested in vivo indicated significant attenuation, spleen, heart, lung and kidney. To confirm the result, some of the scholars isolated lipopolysaccharides mutant and wild-type using silver staining and SDS-PAGE and compwered them (Han, 2014, p. 489). The mutants show alteration banding pattern in comparison to the wild strain. Further, the roles of lipopolysaccharides were associated with systemic infection and bacterial resistance to serum complement (Rendón et al., 2007, 1042). In the recent studies, researchers have found three major mutants, EA10C1, EA16G1 and EA1A1 with mutation genes used in the synthesis of polysialic acid. This gives new insight into the roles of lipopolysaccharides in colonization of chicken, especially, in lungs (Han, 2014, p. 489; (Van Loy, Sokurenko and Moseley, 2002, p. 696).). The finding confirms the importance of these molecules in the pathogenesis of APEC.
Association of Iron Uptake and Survival of Bacteria
Mutants with insertional genes that were either directly or indirectly involved in the iron uptake process were isolated (Dziva and Stevens, 2008, p. 356). Their analysis showed attenuation in distinct tissues. These were involved in sequestration of iron in the host leading to an extremely low concentration in vivo. Genes associated with this process were identified which suggest that acquisition of iron systems related to bacterial virulence, particularly, those that cause septicemia. Genes involve in synthesis of protein for iron uptake were found to have significant similarity with those in Salmonella enterica which were coded by an operon with four members. In turn, these proteins were used to mediate iron transportation which provided evidence of role of the factors in pathogenesis of APEC, especially, in the development of avian colibacillosis (Dziva and Stevens, 2008, p. 356).
Periplasmic and Membrane Proteins
Studies identified several genes with similarity to genes that code for different Periplasmic and membrane proteins. The STM mutants showed attenuation in heart, kidney, heart, liver and spleen. This demonstrated that they harbor disruption genes involved in the synthesis of proteins involved in uptake of microcins (Mellata et al., 2003, p. 494). Genes associated with periplasmic protein were identified which take part in the importation of bacterial cell wall. These were used in mediating the effect of APEC virulence through signal transduction mechanisms affecting the virulence loci.
Influence of metabolism by APEC Genes
According to Li, Laturnus, Ewers and Wieler (2005), bacterial virulence is largely influenced by their adaption to the environment. In the studies involving APEC, several genes have been identified which were associated with metabolic functions. Some of the mutants have been determined: EA11E1, EA7B2, EA1C4 and EA10F11 (McPeake, Smyth and Ball, 2005, p. 245). They contain insertions in the genes involved in the transportation of sugar. These genes were used in pathogenesis process for in vivo survival during upon infection thus influencing APEC virulence. Further, several studies have identified genes associated with encoding of putative desthiobiotin enzyme which was expressed in high quantities during in vivo during infection by the bacteria. At the same time, researchers identified putative pathogenicity islands in the spleen, liver, and lungs. Determination of these genes through competitive assay led to the conclusion that genomic islands in APEC were critical for the pathogenesis of the microbe (McPeake, Smyth and Ball, 2005, p. 245). However, most authors seem to note that further investigation is required to identify the actual functions of these islands.
Function of Adhesins
According to Badger, Wass, Weissman and Kim (2010), adhesins were known to be primary cause of colonization of chicken by facilitating and mediating early infection. Based on the systematic research review conducted, research has identified two major types: type 1 and p fimbriae that were related by pap and fim operons. The STM studies have determined genes involved in encoding type 1 fimbriae regulatory protein. These were identified as factors that initiate infection of host by APEC. Further, these were required for the first step of bacterial infection (Edelstein et al., 2009, p. 836) According to the Badger, Wass, Weissman and Kim (2010), pathogenesis of microbes is a complex process that entails diverse mechanism. Further, use of adhesins is one of the primary strategies that bacteria use to sustain themselves as well as overcome barrier to their survivability presented by the host body systems. The STM result from IMT5155 confirmed that microbial adherence to the surface of the host is by far the critical phase for successful colonization which begins by attachment of the receptors on the mucosa of the host. This is an important step because it contributes to cleaning the layer for penetration into internal organs (Weissman, 2006, p. 975). Therefore, fimbriae and their effects play a vital role in the pathogenesis of APEC infection. The result showed that once the chickens were infected with STM mutants, IMT5155, the complemented form of the bacteria colonized the lungs.
Conclusion
In this paper, it has been imperative to focus on the entire process that occurs in STM using E.coli IMT5155 strain. This is a technique that was identified by Hensel et al. (1995) which become one of the most successful approaches to determining genetic factor in vivo that take part in virulence of the pathogen. In general, it is a form of hybridization method used in collecting transposons while incorporating different sequence of DNA tags which were often a short sequence of 40 base pairs. When these were used to mutate an organism, each mutant can be distinguished theoretically from other mutants based on the distinct tags carried by the transposons in the genome. It involves three major processes: bacterial pooling, tag labeling and hybridization leading to the generation of output and input pools. The last stage allows identification of mutant unable to survive the selective process. One of the advantages of the STM, as noted earlier, is that it helps researchers to study a large number of mutants at the same time. Among the many organisms that have been studied using this approach, it has been imperative to focus on E.coli, a typical colonizer of gastrointestinal tract of warm-blooded animals including avian. It is also one of the organisms that lead to large economic losses in the poultry industry. As such, most studies have tailored their attention to identifying the cause of virulence of E.coli aimed at understanding how to mitigate the problem. Its mechanism of pathogenesis remains unclear, but several virulent factors have been implicated. STM involves several sub-steps important to achieve this goal. The first is the identification of bacteria strain and plasmids as well as materials to use. Most of the research use while leghorn chicken free from specific pathogen. Also, the process entails assembling necessary primers to be used during PCR reaction. As identified the method encompasses generation of APEC mutant library which is attained through bacterial conjugation and generation of antibiotic labeled probe for Southern blot hyb...