Transformation means change. In molecular biology, transformation is a form of genetic change where a cells genetic material is changed through addition of foreign DNA into the genome. Gene transformation is a useful procedure in biology as it is applicable in many areas of biotechnology. Through transformation, genetic traits such as drought and frost resistance can be incorporated into plants. Additionally, transformation helps in cleaning oil spills as bacteria are genetically engineered to digest oil particles. Rapid developments in technology have also facilitated the cure of diseases caused by defective genes.
In this experiment, we will be transforming a bacterium, E. Coli which is found in the human gut. This bacterium was chosen for its relative genetic simplicity as it consists mostly of one large circle of DNA. They also have plasmids which are small loops of DNA with 5000-10000 base pairs. Bacteria can also transfer plasmids back and forth naturally thus allowing them to share genes which plays a major role in their high adaptability.
The first experiment uses a plasmid that encodes the gene for the Green Flourescent Protein (GFP) which comes from Jellyfish Aequorea Victoria. This is the element responsible for the jellyfish ability to glow in the dark (Lacowicz, 2013). After undergoing the transformation procedure, it is expected that the bacteria will acquire and express the jellyfish gene by producing a fluorescent gene that will glow when placed under ultraviolet light. Because of this glowing feature, scientists have long utilized GFP as a reporting molecule. A reporter molecule is attached to the actual molecule of interest so one can follow the molecule of interest by following the colored reporter molecule.
Normally, transformation is done via heat shock in or calcium chloride shock where chemically-competent cells allow DNA penetration after heat shock. However, this method is very inefficient as the typical transformation rate is 1 in 10000 cells while in electroporation, the transformation rate is 1 in 100 cells, thereby making it very efficient. Heatshock therapy is also a delicate procedure since keeping the bacteria in hot mediums for prolonged periods will denature them.
In electroporation, the cells under study are subjected to a high voltage electrical shock that disrupts the cell membrane enough to allow plasmid vectors with the insulin gene to enter the cell. This efficiency is further enhance through the use of the linearized TOPO TA vector, Pcr2.1-TOPO which has a single overhanging deoxythymidine residues at the 3 ends next to the sequence 5-CCCT-3 which allows for base pairing of T and A residues occurring between the PCR products and the vector (Tuschl, Elbashir, and Lendeckel, 2014). Its exact biological role is to cut and rejoin DNA molecules during replication. It specifically recognizes the 5-d(C/T)CCTT-3 parametric sequence and then cleaves the phosphodiester backbone following this sequence.
The second experiment involves gene cloning. This is the process of producing identical copies of a specific gene. In cloning the GFP gene, there are several prerequisites. One needs a source of DNA that harbors the gene under study, restriction enzymes, a vector such as a plasmid to receive the gene of interest and a host to reproduce the gene of interest at an acceptable rate.
Our ligation procedures yielded no colonies on the ligation plates but had multiple colonies in the control experiment. This varied from other students experiments that had colonies in both the ligation and positive plates.
Transformational efficiency is the efficiency with which cells can incorporate extracellular DNA and express the genetic characteristics contained in it. In molecular biology, it is the number of colony forming units that can be produced by transforming 1 ug of plasmid.
Transformation efficiency equationTE = Colonies/ug/Dilution Colonies = the number of colonies counted on the plate ug = the amount of DNA transformed expressed in ug Dilution = the total dilution of the DNA before plating
In this experiment, the number of resulting colonies was 101 CFUs.
Amount of DNA used (ug) = concentration of DNA * Volume of DNA
Transform 6 ul of DNA into
1ul of bacteria the dilute with 250ul of SOC
Dilution = 1/250 * 90 = 0.36
TE = 101.0.
The transformation efficiency for our experiment is a bit on the lower side from the expected figure of 150 CFUs in each plate. There are several factors that may have influenced the transformation efficiency including the composition of the medium, and the composition of the DNA used in the experiment. The composition affects transformation as it carries vital components such as amino acids, vitamins, glucose, and peptide. The amount of salts present in the medium has a great effect on transformation which may be the case with our super optimal broth with catabolite repression. Additionally, the state of the DNA may have had an impact on the end results. Supercoiled DNA enters bacteria easily as compared to linearized DNA. Therefore, our DNA solution may have had some linearized DNA strands that did not transform. In addition to these possible explanations, our genetic strands may have become damaged during the heat shock process.
+ Control Experiment
The positive control experiment allows the researcher to determine whether the experiment is proceeding efficiently. Typically, an experiment should produce approximately 100 colonies. If the insert uptake was successful, the resulting colonies should have a majority of white cells since the control insert DNA reduces the number of blue colonies in the background. The background colonies would appear from an undigested plasmid Vector. The experiment yielded white colonies signifying a success of the transformation process.
However, in some situations, it is possible to find bacterial genes in the blue colonies with the insert of DNA. Similar to the white colonies, the blue colonies also have plasmid but not inserts in the beta-galactoside gene thus making gene uncleaved. The vectors used in an experiment may self-litigate without the addition of an insert which would yield-non recombinant DNA that would create blue colonies. Therefore, additional tests are needed to confirm the presence or absence of the inserts in these genes such as sequencing or Colony PCR.
Restriction Analysis of recombinant Plasmid
The last experiment is a restriction analysis of recombinant plasmid. For this part, we made 3 digests for the PGFPi004 where one digest had Sacll as the restriction enzyme, while the second had Xhol as the restriction enzyme and the last had both enzymes combined. Restriction digesting provides a fast and efficient way of gaining indirect information about a gene sequence. Through this method, the researcher can analyze multiple plasmid constructs simultaneously to determine the presence or absence of an insert, plasmid size, and other site-specific sequence information. This information usually gained by prior investigation of the enzymes properties and characteristics (Nierlich, Rutter, & Fox, 2013). Restriction mapping involves digesting DNA in enzymes and then cutting the resultant DNA fragments through agarose gel electrophoresis. The distance between restriction enzymes is then calculated by examining the fragments produced by the restriction enzyme. In this case, we expected a cut of 4717 bp for the Sacll but the cut was much smaller at 4000bp. Additionally, in the Xhol digest, there were no cuts but a lot of DNA. This may be an indication that the amount of enzymes included in the digest were not enough to cut the gene thus leading to incomplete sequences. The double digest made of Sacll and Xhol was expected to cut the DNA at 4717 bp but looking at the results show that the cuts occur at over 7000bp. This is a clear deviation from the expected results and may be a result of several factors.
Since the movement of DNA fragments is not affected by PH levels in the gel, one of the possible reasons for a deviation in the results is that improper handling during the uv screening stage may have led to the death of some of the enzymes thus making them unable to complete the digestion process.
The lane on the left may also be representative of uncut plasmids as there is a much brighter DNA fragment down the lane. In gel electrophoresis, the migration speed of DNA strands is taken to depend on it size. Thus, larger fragments mover slower as compared to smaller fragments. Because of their reduced size, supercoiled plasmids can move much faster through gel.
Lakowicz, J.R. ed., 2013. Principles of fluorescence spectroscopy. Springer Science & Business Media.
Nierlich, D.P., Rutter, W.J. and Fox, C.F. eds., 2013. Molecular mechanisms in the control of gene expression (Vol. 5). Elsevier.
Tuschl, T., Elbashir, S.M. and Lendeckel, W., MAX-PLANCK-Gesellschaft zur Forderung der Wissenschaften eV, Massachusetts Institute Of Technology, Whitehead Institute For Biomedical Research and University Of Massachusetts, 2014. RNA interference mediating small RNA molecules. U.S. Patent 8,765,930.
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