Through the lens of this experiment, it shows that the gene Rfp encoding, a mutant of the red fluorescent protein – a product of the coral Discosoma striata, AmilCp encoding for the blue chromoprotein from Acropora millespora were duplicated into a pTTQ18 plasmid vector. Accompanied with the amplified PCR of the DNA fragments encoding carrying the Rfp and AmilCp genes, the cloning experiment was initiated. Accompanied with the restriction enzymes Kpn1 and Sma1, both the plasmid and DNA fragments encoding the interest genes were digested. A combination of DNA fragments and DNA ligase were made. The DNA construction plasmid comprising the genes of interest either AmilCp or Rfp went to an evolvement into E. coli strain JM101 cells, paving way of reporter gene and indicating red or blue colour of fluorescent. In order to achieve the appropriate insertion of the reporter gene in pTTQ18, further actions of DNA sequencing were performed.
For effective molecular biology research, one illustration of an essential strategy is DNA cloning. The potential of DNA cloning and plasmid construction use restriction enzymes and ligases to split and reacknowledge DNA fragment and the vector (Svein & Rahmi, 2014). The appearance of diversification of colour diversity in corals is the effect from evolutionary processes. The factor that contributes coloration to corals or sea anemones is the chromoprotein (Miyawaki et al., 2008). The coral Acropora millespora contains a blue colouration due to its two mutation such as the S64C and the S183T (Alieva et al., 2008). The use of any external tools will not be necessary in viewing the pigments since it can be easily seen and identified directly through the naked eye which will be practical as reporter gene proteins. This experiment will have PCR amplified segments of DNA, bearing the AmilCp and Rfp genes. The categorized plasmid vector pTTQ18 and PCR product assisted with restriction enzymes and the expected DNA fragments are put into gel-purification and ligated with the enzyme DNA ligase for the production of a DNA molecule recombinant. The transformation of the recombinant plasmid to E. coli JM101 cells, permitting action of the reporter gene; stimulating fluorescent E. coli colonies in a colour of red and blue. The operation of DNA sequencing was only done with the coral Acropora millespora. This approach was intentionally designed for verification of accurate insertion of the reporter gene in pTTQ18.
In each pair of students, prepare restrictions composing of individualized PTTQ18 and the reporter gene PCR product (separated tubes) accompanied with SmaI & Kpn1 enzymes. The following range of components that are to be mentioned must be provided; 6uL of sterile water, 10uL of DNA (pTTQ18 or reporter gene PCR product), 2uL 10x Kpn1 / Sma1 Buffer, 1uL of KpnL and lastly, 1uL of Sma1. When ingredients are all set, for incubation process, place microfuge tube rack on front bench at a temperature of 37° C for 1 hour. Then export samples from the restriction digest and run through agarose gel. Infuse 4uL 6× loading buffer to 2uL samples before exporting samples to the designated well. (Consciousness of the samples assigned to the lane beforehand is highly encouraged). Then start running the gel at 100v for 30min. Afterwards, demonstration will be done for the DNA visualization using GelRed & how an image is captured of the gel through the ability of an imaging system.
For the ligation process, the paired students will use the samples of the restriction digested process from the previous week to stimulate ligation. An approximate estimated ratio of 3:1 will be vectored that will be handled by insertion. Explanation of the method will be done in classroom. Then with caution, label the tubes and set on lidded pack prepared on front bench. Use the record sheet to fill out your information in the designated box. For incubation process of the samples, place samples at a temperature of 15°C overnight. Reserve samples for the following week.
Now for the final step, the transformation process (DNA cloning & Sequencing). Out of your ligation mix, take 5uL & place on competent E, coli JM101 cells. Blend and occupy on ice on a period of 15 minutes. Then for 1 minute, heat shock cells at 42°C and an addition of 950uL of L-broth. Mix then incubate cells for 30 minutes at 37°C. Then through the samples, plate 50uL to the plates of the nutrient agar ampicillin. The finally, place rack at behind of the class for incubation process. (37°C overnight).
The cloning and transformation of the fluorescent proteins gene AmilCp encoding for the purpose of blue chromoprotein sourced from Acropora millespora and the gene Rfp, which encodes a mutant of the identified red fluorescent protein under the coral Discosoma striata results were achievable. This type of approach can be of great use if arriving to the condition of cloning an individual DNA insert in a plasmid. pTTQ18 is identified as an expression vector owning a multi-cloning site wherein its landmarked downstream from the hard Ptac promoter, and an ampicillin resistance gene for allocation purposes of a selectable marker for the plasmid.
Restriction enzymes and ligases is an operation being used in cloning and plasmid construction for the sole purpose of dismantling and reconnecting DNA fragments. And because of this, it is considered a significant tool in molecular biology (Liljeruhm et al., 2018). The approach is appropriate when dealing with fragments of two. However, is not recommendable due to inefficiency when there is a heightened production of DNA fragments due to the sticky tip accountable for restriction enzymes (Guo et al., 2005). This sort DNA molecules is incapable of supplementing sufficient stabilization or selecting for effective plasmid construction if fragments of 3 or more are being used. In addition, if there is a rise of difficulty due to additional DNA fragments, restriction site in the plasmid by means of conventional cloning show implication on additional subcloning actions that take up a large amount of time, possibly even months just to arrive the conclusive expected outcome of plasmid (Hill & Eaton-Rye, 2014). For the effectivity of life-cell imaging in research, one notable tool that we can put use is the fluorescent proteins. Wherein its potency has initiated visualizing dynamic processes within the cells and putting to convenience in a faster production of new cell imaging strategies. Extended wave light that penetrates tissue effortlessly is the reason why the red fluorescent protein is put to use in 1 vivo imaging (Chudakov et al., 2010).
- Alieva, N.O., Konzen, K.A., Field, S.F., Meleshkevitch, E.A., Hunt, M.E., Beltran-Ramirez, V., Miller, D.J., Wiedenmann, J., Salih, A. and Matz, M.V., 2008. Diversity and evolution of coral fluorescent proteins. PloS one, 3(7).
- Liljeruhm, J., Funk, S.K., Tietscher, S., Edlund, A.D., Jamal, S., Wistrand-Yuen, P., Dyrhage, K., Gynnå, A., Ivermark, K., Lövgren, J. and Törnblom, V., 2018. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of biological engineering, 12(1), p.8.
- Hill, R.E. and Eaton-Rye, J.J., 2014. Plasmid construction by SLIC or sequence and ligation-independent cloning. In DNA Cloning and Assembly Methods (pp. 25-36). Humana Press, Totowa, NJ.
- Chudakov, D.M., Matz, M.V., Lukyanov, S. and Lukyanov, K.A., 2010. Fluorescent proteins and their applications in imaging living cells and tissues. Physiological reviews, 90(3), pp.1103-1163.
- Miyawaki, A., Ando, R., Karasawa, S. and Mizuno, H., Biological Laboratories Co Ltd, RIKEN-Institute of Physical and Chemical Research, 2008. Fluorescent protein and chromoprotein. U.S. Patent 7,345,157.
- Guo, Y.Y., Shi, Z.Y., Fu, X.Z., Chen, J.C., Wu, Q. and Chen, G.Q., 2015. A strategy for enhanced circular DNA construction efficiency based on DNA cyclization after microbial transformation. Microbial cell factories, 14(1), p.18.