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ABSTRACT – Genetic engineering now has straight forward ecofriendly strategies to produce transformation in plasmids. The transformation process allows engineers to introduce DNa with specific desired capabilities into a host plasmid. In this way plants can be created that are able to resist a particular enemy or stressor without having to use toxics like herbicides and insecticides. The bacteria E. Coli was used to determine whether it can be made resistant to ampicillin or not. AmpR was integrated into the host cells and the tracer, GFP was used to identify expressed cells. The laboratory demonstrated that transformation by plasmids so that anti-resistance to the ampicillin can be successfully carried out.
INTRODUCTION
An article from Trends in Plant Science, 2002, welcomed a new “environmentally friendly era in biotechnology. (Daniell et al. 2002 p. 84) Bioengineers can make chloroplast genomes resistant to stresses from extreme weather like drought, from diseases and insects and from herbicides. (Daniell et al. 2002 p. 84) Using plasmids to transform cells is not the only way to carry out a transformation, but it is the eco-friendly way. The ability to perform genetic engineering that can transform chloroplasts has created a branch of ecofriendly strategies to enhance the ability of plants to focus on a particular enemy or stressor without the blanket coverage of toxic chemicals. Daniell (et al. p. 84) notes that the characteristic of being highly polyploid allows the plastic genome “to introduce thousands of copies of foreign genes per plant” after transformation.
Transformation of a cell can occur when a host cell incorporates foreign DNA so that the host cell will have the same capabilities for resistance (or other expression) as the foreign gene. The laboratory introduced a gene into the host, Escherichia coli (E. Coli), that, under the correct circumstances could also exhibit resistance to antibiotic ampicillin. E. coli is suitable because a colony with a population of individual cells can be grown on a Agar petri dish also called an Agar plate (Holtzclaw 2014 Concept 2).
A genetic plasmid is DNA in a circular shape that have genes specific to their unique function; they are not found in main bacterial chromosome. Genetic plasmids have successfully been used in bioengineering to integrate foreign DNA into a bacteria cell and transform the host cell; that is give the host cell the unique capabilities of the introduced DNA. This laboratory used the E. coli cells for the bacteria. The bacteria contained the (+) DNA ampR plasmid that allowed colonies to survive even though the LB agar contained ampicillin. Ampillin is a type of pencillin that acts like an antibiotic and under normal circumstances kills E. coli bacteria.
The purpose of the laboratory was to make recombinant bacteria using E. coli. And then select exclusively for the recombinant bacteria. The resulting colonies for the selection process was viewed under ultraviolet (UV) light to see if it glowed due to GFP (Green Fluorescent Protein). GFP is used to tag the plasmids to identify successful expression.
MATERIALS & METHODS
2 microtubes (for control and experimental cells)
microtube holder
micro pippettes
beakers
4 prepared petri dishes
ice cubes
broth
water (for hot bath)
TE buffer
(+) DNA (pGLO plasmid)
(-) DNA (no plasmid)
100 micro liters of competent cells
The aseptic (clean) technique was used to transfer 10 micro liters of plasmid DNA solution to the microtube labeled (+) DNA and 10 micro liters of TE buffer to the tube labeled (-) DNA. The two tubes were incubated in the ice bath (ice in the beaker) for 15 minutes After the ice path the micro tubes were immediately given heat shock by immersing them in a hot water bath with a temperature of 42◦C for only 45 seconds. And then the microtubes were immediately place in the ice path for 2 minutes.
200 µl of LB broth was placed into each of the microtubes. Then they were placed into a lower heat bath of 37◦C for 30 to 45 minutes. This is the recovery period. During the recovery period 50 µl of E. Coli (+) DNA suspension was spread on the appropriate two agar plates using the aseptic technique with a sterile loop and spreading the bacteria evenly over the whole plate. And then the control plates were prepared by transferring 50 µl of E. Coli (-) DNA suspension using the aseptic technique and spreading the bacteria evenly over the surface of the plate. All the plates were allowed to rest for 2 minutes (sit undisturbed) in order to allow the bacterial media to become completely absorbed by the agar media. At this point the covers were put on the plates and the plates were invert and let to incubate overnight at 37◦C.
Note: During the first ice bath the following plates were picked up 1 LB, 2 LB with Ampicilin and 1 LB with Ampicillin and Arabinose.
RESULTS
Two gel plates were used as control environments. They are picture in Figure 1 & 2. Notice that very little growth is evident on either of the control cell plates, LB or LB Amp. The drawings of the experimental cells with plasmids, on the other hand, Figures 3 and 4, are well populated in comparison. The Arabinose population is higher in Figure 3 (LB gel plate) with a count of approximately 850 while Figure 4 (LB Amp gel plate) has a count of approximately 299. (See table 1)
Figure 1Control Cells LB
Figure 2 Control Cells LB Amp
Figure 3 Exp. Cells with Plasmids: LB Amp plus Arabinose
Figure 4 Exp. Cells with Plasmids: LB Amp
Equation 1 shows the calculation for Transformation Efficiency was found to be approximately 13.0 x 10^3 CFU/µg of pGLO. Transformed populations were large even though very small quantities (in the microliter range) were used. This was possible because commercial prepared cells were used.(Note: CFU/
(50 CFU)(0.4 µg pGlO)260 CFU=0.0769 µg of pGLO 1000 CFU0.0769 µg= 3.0 x 10^3 CFU/µg of pGLOEqn. 1
1. A cell is competent when it has the ability to ‘take up’ exogenous DNA so that it will be transformed. (Griffiths, et al. 2000: Glossary) A cell is competent when it has the ability to ‘take up’ exogenous DNA so that it will be transformed.(Griffiths, et al. 2000: Glossary)The three features that make the E. Coli cells more competent to ‘pick up’ the plasmid DNA are (a) an origin of replication (ORI), (b) a drug resistant marker, AmpR, and (c) high gene density.(Kumar & Garg 39-40) Other features that make E. Coli suitable include the ability to speedily reproduce; colonies are made up of millions of cells in a short period (about 12 hours); and it has no nuclear envelope (or wall) around the bacterial chromosome. One chromosome of E. coli has all the genes needed for reproduction and the basics of survival. (Holtzclaw 2014: Concept 2)
2. The three features that make the E. Coli cells more competent to ‘pick up’ the plasmid DNA are (a) an origin of replication (OR), (b) a drug resistant marker, AmpR, and (c) high gene density. (Kumar & Garg. 2005: 39-40)
3. No growth was expected on the plate with control cells and LB Agar because it did not contain Ampicillin.
Ampicillin is resistant to E. coli so it would not grow on the LB Amp Agar plates.
4. The pGLO causes a transformation of Ampicillin that creates antibiotic resistance, AmpR. The AmpR gene is necessary because when it is carried on plasmid DNA, beta-lactamase enzyme can then be made by the bacterium. The experimental cells with plasmid DNA were added to agar with Ampicillin to see if the plasmid DNA could overcome the E. Coli. It is interesting because if the plasmid DNA is incorporated by the bacteria, the bacteria will become ampicillin resistant
5. The colonies growing on the plates with Ampicillin and containing arabinose sugar should glow when placed under Ultraviolet light.
6. The purpose of the Arabinose is to activate the EGFP gene on the plasmid used in the bacterial cells.
7. Below the functional products of the different genes found on the pGLO plasmid are listed.
Ara C – Codes for a regulatory protein that binds with the providers.
Beta-lactamase – Codes for a protein enzyme produced that allows bacteria to inactivate penicillin (or in this case Ampicillin) and become resistant to it.
EFGP – coded for glowing: green fluorescent (material from jelly fish causes the fluorescence).
Conclusion
The variation in the number of colonies between student projects could be due to contamination from the air or from the transformation tubes is the tubes were not perfectly clean. In fact every step has to use tools that have been purified, such as the sterile loop used when inoculating the media. The variation could also be due to contamination from the air or from the transformation tubes is they were not perfectly clean. The laboratory demonstrated transformation by plasmids so that anti-resistance to the ampicillin was created.
REFERENCES
Daniell, Henry, Khan, Muhammad S. and Lori Allison. (2002). Milestones in chloroplast genetic engineering: An environmentally friendly era in biotechnology. Trends Plant Sci., 7(2), 84-91. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476055/
Griffiths,Anthony J.F., Miller, Jeffery H., Suzuki, David T., Lewontin, Richard C. and William M. Gelbart. An Introduction to Genetic Analysis. 7th Edition. New York: W.H. Freeman and Company. 2000. http://www.ncbi.nlm.nih.gov/books/NBK21993/
Holtzclaw, Theresa Knapp. Bacterial Transformation. The Biology Place, Pearson Education, 2014.http://www.phschool.com/science/biology_place/labbench/lab6/intro.html
Kumar, Anil and Neha Garg. Genetic Engineering. Nova Scotia: Nova Science Publishers, Inc. 2005.
