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Introduction: Site-directed mutagenesis enables researchers to inspect the behavior and sequence changes or analyze a range of mutants to attain desired changes. There is connection between the three genes that is; I1, I2 and O2 and interaction between AraC proteins. The small DNA chronology is identified by AraC protein. In this study the genes chronology and nucleoside is changed at four strategic sites in order to find out the effect of mutation on transcription of the AraC locus, in-vitro.

Results and Discussion: Transformations were positive and this is because they did not show any problem with mutagenesis reaction. Several numbers of colonies were transformed. The transformed colonies directed mutagens. Every colony is a culmination of site directed mutagenesis reaction coming from a single recombinant DNA molecule having undergone division via cloning.

Limitation: A 10% reduction on the promoter activity meant that no change would be observed. Moreover, there was failure to extract DNA in the sequencing process and this means that further investigations on mutations regions would have been present.

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(Key words: SDM, mutation, PCR, GFP, pGlo, DNA, RNA)

Introduction

According to Sabel & Brookman (2012), “Site-directed mutagenesis is an in vitro method for creating a specific mutation in a known sequence, and is typically performed using PCR-based methods.” Primers used in the investigation of the behaviors of mutations have the ability to of making small sequence changes and as such, extension of the primers or inverse polymerase chain reaction, (PCR) would at times be used to extend the regions under mutation (Reikofski & Tao 1992). Site-directed mutagenesis, (SDM) procedures enable researchers to examine the behavior and sequence changes or analyze a range of mutants in a bid to address the mutation changes as desired. For instance, the use of molecular genetics helps in investigating the functioning of genes. This is because the previous procedures like use of radiation either do not give more specific details or could cause harm to the organisms. The use of radiation is random and may not target specific region of the DNA molecule, as the procedure does not recognize gene sequence.

The statement above calls for the development of other SDM procedures that target specific gene sequence. One example by Blank, Henkel & Gerlach (2011) is “Homologous recombination implies technologies of gene targeting, an introduction process of recombinant DNA into specific sites mediated by endogenous recombination machinery, and recombineering which refers to the engineering of recombinant DNA,” (p.e15763) supported by (Zoller 1991). This class experiment aims at investigating the gene functions using molecular genetics. In this procedure, a number of genes (M2, M4, M7, and M8) are modifiable to attain an extended region of DNA sequence. As noted above, PCR can be used to modify gene sequence. In addition, restriction of enzymes can also be used. This research employs the use of arabinose locus in E-coli in studying the SDM procedures, which is a typical prokaryotic method. The locus AraA, AraB, and AraC are grouped in a single genome in a RNA messenger before being transformed to three different forms of protein. This according to should be at “37%u2005°C to induce intra-genomic recombination.”  

It is important that the prokaryotes and eukaryotes should be expressed appropriately and in timely manner by monitoring time sequences via the on and off buttons when the genes show different expressions (Ho, Hunt & Horton 1989 and Zalieckas, Wray & Fisher 1998). The following are needed in the study, AraC gene chronology of the supporter, RNA polymerase is added, and manufactures of protein dimer, 2 pair of DNA chronology that connects to 2 sequences, the O2 s sequence and the I1 sequence that makes a DNA loop. The loop pattern stops RNA polymerase from coming close to the promoter areas, concurrently changing of the gene. Upstream of the promoter are 3 sequences, O2, I1 and I2 series that are capable of binding the AraC protein, controlling the switching on and off of the transcription. In the existence of Arabinose, the confirmation of protein changes to open up the loop arrangement. The freed AraC sequence connected to I1 and I2 areas, enhancing RNA polymerase to bind to the promoter and enabling transcription to take place. According to Manuel, Costa & Sa-Noguiera (2003), “it should be possible to detect an RNA transcript stopping at cre araB due to its location far downstream of the promoter region.” It is therefore the conformational change of the dimer that controls the binding of arabinose. Thus during arabinose, carbon source, existence of metabolism is promoted, during the presence of a carbon source.

There is connection between the three genes that is; I1, I2 and O2 and interaction between AraC proteins. The small DNA chronology is identified by AraC protein. In this study the genes chronology and nucleoside is changed at four strategic sites so as to find out the effect of mutation on transcription of the AraC locus, in-vitro. The targeted progression has a pBAD promoter, followed by O2, I1 and I2 genes. These DNA sequences were removed and placed into a plasmid, to determine the effect of mutation in these DNA sequences, a fluctuation in protein production. This transcription movement can be analyzed by a reporter gene GFP fluorescent protein. In the absence of arabinose, pGlo does not shine. The existence of arabinose makes pGlo to fluoresce within UV light. A plasmid containing the targeted gene mutation is incorporated into a plasmid that is taken up by E-coli bacteria.

Materials and Methods

Procedure of Genetic Mutations

By the use  bacterial pGlo plasmids, a double stranded DNA, was used  as a template in the chain reaction, mutations was  aimed at areas, I1, I2, O2 and a mutation of  GFP protein that stopped  shinning. Plasmid mutation copies were formed by using PCR.

The chain reaction of Polymerase was performed by the use of small levels of reactants. After PCR has been performed it was left to stay overnight in a thermal cycler. The preparation of agarose was organized for the analysis of reaction the day after.

The analysis of the reaction took place on agarose gel. 25microl, 12 microl were taken away for further examination on gel to view fluorescence on an agarose gell. The remainder agarose was then stored for later use.  Then 3microl of loading die were added to monitor electrophoresis.

Transformation and plating clones on plates of agorose gel. Reagents were kept on ice all time. The other 13 microl, 2 types of DNA molecules were also included. The original Pglo template and the product of the PCR containing site were directed mutagenesis. By the use of heat shock and transformation of the plasmid DNA were introduced into E-coli bacteria, and then was followed by a screening of antibiotic resistance.

AraB AD Locus in E-coli

The encoded genes AraA, AraA, and AraC and other products of gene were involved in Arabinose on the inner and outer parts of the cell respectively. The AraA, AraB, and AraC genes were transcribed using Pbad along biochemical pathway. The Pbad promoter was bound by RNA polymerase to boost initial transcription. The practical investigated changes made the SDM process to determine the gene functions.

The M8 mutation targeted the GFP protein region in order to deactivate the protein and this showed that the M8 was used as a control mechanism in the SDM process. The original Pglow was isolated from E-coli and were mutated using forward and backward primers. The result was the production of two distinct molecules: one being the original pglow DNA sequences and the other was circular DNA mutations. During transformation of the template mixes, an experimental error led to over production of original plasmid. The activity was redone in to produce adequate unmethylated DNA templates. The next procedure was the transformation of digested Dpn1 templates into E-coli that led to the formation of trasformanants with site directed mutants.

The D Protocol

C residue was reduced to T residue in order to test expression of the M2 mutation in the locus. Similarly, M4 mutagenesis, M7 mutations, and M8 mutations were expressed via different procedures.

Results and Discussion

Transformations were successful. This is because they did not show any problem with mutagenesis reaction. Several numbers of colonies were transformed. The transformed colonies directed mutagens. Every colony is a culmination of site directed mutagenesis reaction coming from a single recombinant DNA molecule having undergone division via cloning.

To enhance the probability of transformant having the mutant under experiment to compare how it behaves to wild type Pglow in the presence or absence of arabinose. From the last streaked and grown colonies each selected colony was divided on plates containing arabinose or lacking arabinose. Further investigation of the GFP expression under UV light would draw conclusions about the effect of single or double nucleoside mutations on control of transcription in selected colonies. To recover single clones and test sufficient amount of bacteria on medium that either contains or and lacks arabinose.

Final part of the experiment l, scoring to observe the effects of each mutation to the template. It is found that under UV light plate with no arabinose should be without fluorescence and no GFP expression. A plate which contains arabinose should have light due to GFP presence. Some GFP light from wild type Pglow existed on arabinose lacking plates.

Four different mutations were done to targeting different areas in the sequence of genetics. Nonetheless, the experimental set up was limited in that the DNA did not come from the trasformanants and as such, there was difficulty in sequencing them to letting them show the mutation points upon introduction. This means that M8 was the only important gene in the mutation. Translation M4 gene resulted to AUG codon translation that finally translated to amino acid to produce a truncated GFP protein, which never fluorescent. On arabinose plate there was no fluorescence apart from two signifying that DNA had a residual effect indicating that it was a wild template type.   

The sequence motive for I1, I2, and O2 are binding DNA target in the case of Pglow plasmid in a AraC protein. Without arabinose, the AraC dimer the next sequence of AraC binds with O2 but not I2 because the new loop formed does not permeate RNA polymerase to flow into the promoter (Miwa & Fujita 2001).

There was a demonstration of strong GFP expression phenotype when arabinose was introduced but weak expression of the same without GFP arabinose. This is because of loop disintegration necessitating the polymersa to bind and encourage transcription. In addition, this could have been because of formation of a weak loop with 2 2 sequences that enables RNA to allow polymerase to transcript. This does not allow the formation of the loop. It can be reported that GFP is a very alert reporter upon formation of a protein. This implies that the RNA products like the prokaryotes shows a very strong correlation between successful transcription and the how strong the promoter is.

Limitations of the Research

The quantitative data presented in the report were not so accurate and this implies that even though this was a quantitative analysis, it can also be best described as a qualitative test. The various GFP expressions in the mutagens imply that there was no accuracy in measuring the activities of the promoter. A 10% reduction on the promoter activity meant that no change would be observed. Moreover, there was failure to extract DNA in the sequencing process and this means that further investigations on mutations regions would have been present.