Analysis that this mutation causes missense mutation

Analysis of knockout mutant E.coli strainargG under anaerobic and aerobic conditions.

Authors: Ademutola Akinnuoye, RamouFofana, Pratibha Gurung, and Sanchi Rani    The group was expected to decide on one strain to study bacterial virulencefactors of E.coli, using D. discoideum as a model. D.

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discoideum was chosen as a modelbecause the organism has advantages in that it is a genetically tractablehaploid eukaryotic organism with accessible phenotypes.  The strain was chosen by identifying whichmutant strain was observed the most resistant to phagocytosis by D.discoideum. This was sequenced usingan Illumina MiSeq.

After screening the four different strains, the groupdecided on strain MG 1655. This mutant was identified as ArgininosuccinateSynthase (argG). Using bioinformatics analysis, the group found that thismutation causes missense mutation (Asp-Asn). It was also found that this mutation was involved in synthesizingcreatine, polyamines, arginine, urea, and nitric oxide.   The research began by analyzing thedifference of growth between WT and argG in an Iron Scavenging Assay. To haveclear results showing the difference in cell colonies, WT and argG were dilutedfour times.

The results indicated that argG had les growth than WT. Thisindicates that argG survives poorly in low iron environments.  To further investigate, the group wanted tocompare the growth of argG under aerobic and anaerobic conditions. Again, thisexperiment utilized the Iron Scavenging Assay. This experiment utilized a M9plate, LB plate and LB supplemented with sodium nitrate ( at concentration5mM). Like the previous experiment, there were a series of dilutions to comparethe differences in growth. The final results were that argG- deficient bacteriagrew poorly in M9 media and LB supplemented with sodium nitrate. This indicatesthat argG may play a role in nitric oxide in the phagosome.

In conclusion,based on the results of the experiments, the mutant strain argG may play a rolein metal binding protein that is involved in nitrate reduction. By knocking outargG, increased levels of nitric oxide will be inhibited, resulting is lesskilling of bacteria through nitric oxide.     I learned that virulence factors allows bacteria to either invade itshost, cause disease, or evade from the host’s defense mechanism. By firstidentifying these virulence factors in the selected mutant strain, we will beable to understand how evasion of D. discoideum predation may contributeto coincidental evolution of virulence factors against mammalian hosts. Bymanipulating the environments (plates), you can observe the changes in growth/survival of argG. With enough trials, researchers can create a list ofdifferent characteristics that allow argG to survive phagocytosis by D.

discoideum. They can then characterize other E.coli mutant strains to identifywhich common traits allow survival.  Analysis of PgaB mutation in EscherichiacoliAuthors: Manyi Foncham, ChristineHillman, Michelle Bomgardner, and Zairah ShahidIn the beginning of the semester, eachmember of the group was given a different mutant strain of E.coli. The group was expected to decide on one strain to studybacterial virulence factors of E.coli,using D.

discoideum as a model. D. discoideum was chosen as a modelbecause the organism has advantages in that it is a genetically tractablehaploid eukaryotic organism with accessible phenotypes.  The strain was chosen by identifying whichmutant strain was observed the most resistant to phagocytosis by D.discoideum. The group decided onstrain pgaB by plating E.coli WT and pgaB and observed the changes in survival.The results of the experiment indicated that pgaB was observed to be the leastresistant to phagocytosis by D.

discoideumout of the four strains. Utilizing Bioinformatics, pgaB was found to causenonsense mutations, changing cytosine to thymine.  It was also indicated that pgaB is involvedin a four gene operon to generate and maintain a biofilm formation process as aprotective defense utilizing extracellular secretions. The first experimentused a Microliter Dish Biofilm Formation Assay. This assay was used to measurethe formation of biofilm.

The biofilm was then stained with 0.1% crystalviolet. The results showed the decrease in biofilm formation in pgaB comparedto WT.  The results also indicated abiofilm formation decreased in both WT and pgaB when pH decreased. It was alsoobserved that biofilm formation slightly decreased in an iron deficientenvironment. The group furthered their investigation by using an AntibioticResistance Assay. The results showed a decrease in biofilm formation whentreated with liquid antibiotic Ampicillin.

    I learned that Microliter Plate biofilm assay is a useful way thatresearchers can analyze bacterial attachment by measuring the staining of theadherent biomass. I was unfamiliar with this assay but the presenter explainedthat biofilm is a naturally occurring process of bacteria sticking to eachother. This is a response to external stimuli as a protective defense. PGA isrequired for this process to occur. PGA forms the biofilm molecule.

PGA issynthesized in bacteria then exported out of the cell. This method caught myattention because being in this lab group’s class, Professor Snyder provided usthe basic assays that we could work from but also gave us the opportunity toresearch new methods. I’m impressed how well the experiment worked to provetheir hypothesis.  The Genetics of Triodopsis: SequencingCytochrome C oxidase I to Determine SpeciationAuthors: Jimmy HenrichsPolygyride, a family of air breathing land snails,are believed to date back to when North America split from Africa. This eventwas about 145 million years ago. Trodopsisare dated back 125 million years ago. This genus is believed to be the resultof the mid-continental seaway incursion. There are approximately 1000 differentspecies of land breathing snails in North America, resulting from geologicalchanges.

You can determine if two land snails are either separate species, thesame species, or in form of sub speciation by genetic sequencing ormorphological comparison. In this experiment, Cytochrome oxidase I wassequenced between four species of Trodopsis.These four species include: complanata,fraudulenta,tridentate and tennesseensis. Cytochrome oxidase I was chosenas the mitochondrial gene because it has high levels of sequence such as5.

8S-ITS2-28S rRNA. The experiment consisted of four methods: DNA extraction,PCR and gel electrophoresis, DNA purification and DNA sequencing. In DNAextraction, a sample from the foot of the snail was used in theextraction.  In PCR and gelelectrophoresis, specific primers were used for CO1. DNA was exponentiallyincreased by PCR and was ran on an agar gel. In DNA purification, the sampleswere cut from the gel then purified using a Qiagen Gel extraction Kit. In DNAsequencing, the purified samples were then sequenced at Eton Bioscience, Inc.The results indicated that there were nucleotide differences; however, it wasnot consistent through the four species in subject.

For example, there are manylocations through out the sequence where tennesseensis1 shares more nucleotide sequences that are more similar to complanata samples rather than tennesseensis. This may indicate that tennesseensis 1 may not even be a tennesseensis at all and may have beenincorrectly named in the past. Another peculiar incidence showed that Tridenta 2 and tennesseensis 2 have more similar nucleotides between each other,rather than their own species.

Fraudulenta2 was found to share more nucleotide similarities to tridentata1,3 and 4., suggesting that may be related.     I learned that these results of one species fitting into a differentspecies more may be because the species were mislabeled by the Museum ofNatural History or these species might be a result of sub-speciation. Sub-speciation is the intermediate between two different species. Because thesesnails date back over 100 million years ago, this hypothesis is believable andcould be due to evolution. I was also told that another reason for thesefindings is the CO1 gene isnt quantifying genetic differences in Triodopsisconsistently. This could also be a strong possibility due to the fact that CO1gene was found to be inadequate in another experiment.



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