Transformation of E.Coli Bacteria Using Green Fluorescent Protein: 832766

Transformation of E.Coli  Bacteria Using Green Fluorescent Protein

Abstract

Transformation is an important property in molecular biology as it is used to modify the structure of simple organisms such as the bacteria and fungi microbes. The transformation process introduces a new gene in the organism that alters the characteristics of this organism. Most of the times the added traits are for survival purposes of the organism. A bacterium that cannot survive in an environment with competitive fungi, the bacteria can release toxins that kill the fungi. The green fluorescent protein that was recently discovered is contained in the plasmid reporter protein and hence with its fluorescent properties can be used to identify very many other processes occurring in the organisms. In a living cell, it can be used to identify the micro tubules activities. It can also be used to give information on how the bacteria transport the information of releasing toxins when they are in a competitive environment. Immobilized metal affinity chromatography was used to purify the green fluorescent protein that was sub-cloned into the pQE30 expression vector. According to (Jose de melo Silva, Alvas & Pakay, 2016), the purification of the protein was seen through by the attachment of N-terminal protein to hexastidine therefore achieving a 3-fold purification of the green fluorescent protein. Knowledge on the important methods used in molecular cloning, recombinant protein expression and purification have been gained by the students by the end of the practical.

Transformation Of E.Coli  Bacteria Using Green Fluorescent Protein.

Alteration caused by the introduction of a new DNA plasmid to a cell of an organism resulting in exchange of genetic information is called transformation in molecular biology. The foreign plasmid may be obtained from other species that are not related to the organism. Transformation is frequently carried out for simple organisms such as bacteria and fungi. The process is hard with complex multicellular organisms such as plants and animals. This is because those organisms require alteration of numerous cells which is difficult to do.

According to (Zou, 2014), molecular biology and genetic engineering improvements involve the vital use of cell transformation through the introduction of a new plasmid into a microbe such as a bacterium, which then rapidly multiplies the plasmid to make a very huge amount of it. According to (APS, 2018), a tiny round piece of DNA containing genetic information responsible for the growth of bacteria is what is called the plasmid. The plasmids are used by the bacteria to share information among themselves in order for them to produce toxins that are used against other microbes growing in the same niche such as fungi and molds. The plasmid is therefore important for the bacteria`s survival.

 According to (Tsien, n.d.) organisms normally reject new substances introduced in their bodies. Bacteria must therefore be made to accept the DNA with the plasmid. This is done by creation of holes on the cells of the bacteria by suspending them in a highly concentrated solution of calcium. The DNA is then introduced into the bacteria (APS, 2018). The E.coli bacteria is normally used in these experiments. Green fluorescent protein and protein coding for ampicillin resistance are introduced to the E.coli as they are present in the plasmid. According to (APS, 2018), the transformed E.coli from the experiment that have changed through their ability to survive in a niche with antibiotic ampicillin are determined through the ampicillin resistance gene.

The E.coli used for this experiment are not considered pathogenic as they are part of the bacterial flora in the human gut. The aims of this experiment were to comprehend recombinant DNA techniques such as the transformation procedure. Also gaining the knowledge on the function of the reporter gene green fluorescent protein in screening for the gene of interest was of vital importance. Carrying out the transfer of DNAto another organism changing the traits was an important purpose. Lastly, understanding the sterile and decontamination methods used while handling bacteria was vital.

Figure 1 and 2 showing cell transformation.

Materials and Procedure.

. 24.25 microlitres of sterile water was added to one of the tubes. Afterwards, to this same tube, 5μl of 10x vent buffer, 5 μl of PE-GFP template, 5 μl primer E1, 5 μl primer E2, 5μl of 2.5Mm dNTPs and 0.75 μl of Vent polymerase were added simultaneously to the test tube with fresh sterile tips used for each addition of the reagents. The samples were then placed into the PCR and run for 2 hours and 45 minutes. While the PCR ran, agarose gel was prepared After the PCR run time was over, the PCR tube was collected and the PCR sample was all of it transferred to a sterile microfuge tube that was labelled Tube 1. The original PCR tube was then rinsed with 150 μl of sterile water and this fluid was transferred to Tube 1. The contents were then mixed. 20 μl of the diluted PCR from Tube 1 was transferred to another microfuge tube labelled Tube A. 5 μl of 6x DNA sample leading buffer was added to Tube A and the contents were mixed gently followed by spinning the tube for 10 seconds. The sample was then added to the agarose gel and the lanes where the gel was loaded was recorded. The lid was then attached to an electrophoresis apparatus and the gel was run at 120 volts in the apparatus for 30 minutes. Afterwards, the agarose gel was transferred to the UV trans illuminator to visualize the DNA bands. 180 μl of membrane binding solution was added to the remaining 180 μl of diluted PCR in Tube 1. One SV Minicolumn was placed in a collection tube. The prepared PCR product in Tube 1 was then transferred to the SV Minicolumn and were incubated for 1 minute at room temperature. Afterwards, the SV minicolumn was centrifuged for 1 minute and the liquid formed in the collection tube was discarded. The column was returned to the collection tube and was washed by adding 700 μl of membrane wash solution and centrifuged again for 1 minute. The collection tube was emptied and the washing process was repeated as well as the centrifugation. Afterwards, the SV minicolumn was transferred to a clean 1.5 ml micro centrifuge tube and 20 μl of nuclease-free water was added directly to the center of the column. This was then incubated at room temperature for 1 minute. Thereafter the SV minicolumn was discarded and the micro centrifuge with eluted PCR was stored.

150ml of distilled water was added to a 250ml flask. Thereafter, 1.5g Bacto tryptone, 0.75g Bacto yeast extract and 1.5g NaCl were added to the flask and the flask was swirled to dissolve the contents. 2.25g Bacto-agar was then added. The balance and surrounding area was thoroughly cleaned with moist tissue. The neck of the flask was then covered with non-absorbent cotton wool and 3cm of autoclave tape was attached. The mixture was then autoclaved. In another flask, 150ml of distilled water was added. 1.5g Bacto tryptone, 0.75g Bacto yeast extract and 1.5g NaCl were added to the flask and the flask was swirled to dissolve the contents. The balance and the surrounding area was thoroughly cleaned. The neck of this flask was also covered and sterilized in an autoclave. The PCR product dissolved in GFP DNA samples was put in a tube labelled Tube 1. 1 μg of Pqe30 vector was cut and put in 18 μl of sterile water in a tube labelled Tube 2.18 μl of sterile water, 2 μl 10x BufferE and 5 μl Bam Hi premix was added to tube 1 and to Tube 2, 2 μl 10x BufferE and 5 μl Bam Hi premix was added. The reagents in each tube were mixed gently and the digestions were allowed to take place for 2 hours at 37 degrees Celsius. Afterwards, agarose gel was prepared. To each of the digested tubes 175 μl of MilliQ was added followed by addition of 200 μl membrane binding solution and were mixed. The purification of the digested GFP DNA was done using the SV Mini Columns as previously done. In the quantification of the digested DNA, Nandrop spectrophotometer was used. The concentration of the DNA was then recorded. Afterwards ligation of the GFP DNA was done followed by the analysis of the DNA digests using agarose electrophoresis. Preparation of E.coli cells followed and thereafter sub-cloning whereby the bulk of the induced cells were harvested and stored until purification by IMAC was done. The LB was prepared to give a final concentration of 100 μg/ml. 300 μl of TENS buffer was added, 150 μl of 3M sodium acetate was also added and finally 0.9ml of ice cold absolute ethanol was added and all were mixed at each interval. 200 μl of 70% ethanol was used to wash the DNA pellets. Each pellet was then resuspended in 50 μl of TE. The plasmid DNA was then dissolved in 25 μl of TE/RNase solution. Immunofluorescence microscopy procedure proceeded afterwards. The samples underwent processing before the analysis using SDS-PAGE. 50ml of the cells were used to purify hexahistidine-tagged GFP through the IMAC method. After the purification of the hexahistidine-tagged GFP. Induction time samples were then treated before the addition of SDS-PAGE using loading buffer that was equal to the cell densities of the suspensions. The induction GFP purification samples were then done for the purification using SDS-PAGE. Cloning and confirmation and GFP Induction proceeded and lastly, was the purification of GFP by IMAC method. Results were recorded in each step.

Results

Practical Image 1:

Smearing is observed for the PCR assay obtained. An extra band is also observed for this result. Other results are in accordance with PCR assay expected.

Practical 2 Results.

Practical 4 Results.

Smearing is observed in the PCR assay for the sample obtained.

 Practical 5 Results.

The results show the band lengths formed by the DNA samples as they travel towards the anode due to the presence of the phosphate groups present in the DNA. The solutions were allowed to run for a long period of time in order to allow the bonds to separate in a distinct way. A DNA ladder is loaded as a standard in order for it to be used to know the length of the original samples. The smallest fragments travel the furthest because they are easy to push with the electric field. The agarose gel used was for separation of big pieces of DNA.

 Standard Curve for the results of practical 4.

Distance migrated                      DNA Size

 Discussion.

Multiple colonies of bacteria had a whitish color as observed from the first control plate. The bacteria represented the full transformed cells that had fully absorbed the plasmid that inserted the ampicillin resistance gene that boosts the survival of the E.coli on the plates that contained the ampicillin (Allison & Gilbert, n.d.). The second control plate`s bacteria only turned white when illuminated with normal light. Minus the light, the bacteria had not turned white. However, an exposure to ultra violet light, the cells fluoresce a green light color indicating their transformation. These cells were also having the ampicillin resistant gene that enhanced their survival. Arabinose sugar present in the agarose was useful as it activated the expression of the green fluorescent protein gene and hence, only this plate glowed. The activation by the arabinose sugar was through coupling of green fluorescent protein gene and a regulatory protein called araC which occurs in the PBAD protector. For the fluorescent to occur, ultra violet light is necessary to cause the foreign green fluorescent protein in the bacteria to fluoresce (Prendergast, n.d.). The third control plate lacked any microbes because the bacteria that were initially required for the reaction were killed by the ampicillin. The fourth plate had a huge amount of bacteria indicating that the bacteria grew without influence from DNA, ampicillin and other factors. A growth curve 2drawn for these data would result in a similar curve for each isogenic pair because the incorporation of the green fluorescent protein in the bacteria does not affect the growth rate of the organism`s cells.

Extra bands formed on the PCR assays and the bands would not have been formed with an increase in tm of the primer in order to prevent the formation of the extra bands on the PCR assay as observed in the results. A reduction in the template can also be done in order to prevent the formation of these extra bands (Andrews et al 2006). There were smeared bands in the agarose gel electrophoresis for plasmid DNA and this could be attributed to a problem with the running buffer. The concentration of the buffer was not exactly the same as that of the running buffer. Also too much DNA may have been present resulting in the smearing. Reducing the quantity of the sample and reducing the contamination of the DNA will reduce smearing.

Conclusion.

The recombinant Deoxyribonucleic Acid techniques like the transformation procedure were fully comprehended in the experiment provided as this was the method used to incorporate the plasmid into the cell of the E.coli. The plasmid protein was found to be a very important component of the deoxyribonucleic acid containing ampicillin resistance gene and green fluorescent protein gene. This protein was found to be a reporter protein responsible for transmission of information within the cell of the organism. The incorporated new protein was found to be important to the organism as it enhanced its survival in the niche with ampicillin which is a toxin to bacteria. The bacteria that were not incorporated with the plasmid did not survive in the environment and were killed and hence, no bacteria were not observed. The green fluorescent protein which emits green light was seen to be important as it helps in identifying processes that cannot be seen normally such as the activity of the micro tubules of the living cells. All objectives of this experiment were met and therefore, despite the minimal errors observed in the experiment, the experiment was a total success.

References.

American Pytopathological Society.(2018). Transformation of E.coli using Green Fluorescent Protein. APS.  Available from, https://www.apsnet.org/EDCENTER/K-12/TEACHERSGUIDE/PLANTBIOTECHNOLOGY/Pages/Activity4.aspx

Allison, D & Gilbert, P. (n.d.). Modification by surface association of antimicrobial susceptibility of bacterial populations. J ind Microbial. Available from, https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2672.2006.03243.x

Andrews, J.S., Mason, P., Thompson, L. Stephens, G. & Markx, G. (2006). Construction of Artificially Structured Microbial Consortia (ASMC) using Dielectrophoresis: Examining Bacterial Interactions via Metabolic Intermediates Within Environmental Biofilms. J Microbial Methods. Available from, https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2672.2006.03243.x

Jose de Melo Silva, Alves, L.L. & Pakay, J. (2016). Green Fluorescent Protein Purification Through Immobilized Metal Affinity Chromatography and Its Relevance for Biomedical Science Students during Biochemistry Practical Classes. Journal of Biochemical Education. Available from, http://bioquimica.org.br/revista/ojs/index.php/REB/article/download/638/566

Zou, Y. (2014). Green Fluorescent Protein. The Embryo Project Encyclopedia. Available from, https://embryo.asu.edu/pages/green-fluorescent-protein

Prendergast FG. (n.d.). Biophysics of the green fluorescent protein. Methods Cell Biol. Available from, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875081/

Tsien RY.  (n.d.). The green fluorescent protein. Annu. Rev. Biochem. Available from, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875081/