Team:Utah State/Results/Chlorophylasse

Mechanism

Chlorophyll is a photopigment found in the chloroplast of green plants. Chlorophyll absorbs light strongly in the blue and red regions of the visible spectrum. It absorbs very poorly in the green region, and that light is reflected, hence, making plants green. Chlorophyll is an excellent photoreceptor because it contains networks of alternating single and double bonds. Because, the electrons are not held tightly to a particular atom and can resonate, they can be readily excited by light. There are two varieties of chlorophyll molecules, chlorophyll a and chlorophyll b. They have small differences in absorbance spectrums allowing the plant to absorb and utilize more of the sun’s energy. The power of chlorophyll is its ability, when excited, to transfer an electron to an acceptor creating a separation of charge and chemical potential. Thus, light energy is converted into chemical energy (Tymoczko et. al., 2010).

Results

Chlorophyllase Generator Construction

Protein generator part for chlorophyllase. The wheat (Triticum aestivum) chlorophyllase gene was kindly provided to us by Dr. Joseph Jez, Washington University, St. Louis, MO. Primers were designed to use polymerase chain reaction (PCR) to amplify the TaCHL open reading frame from the pET28a vector that the gene was provided in. Since we were using the chlorophyllase gene in gene fusion applications, RFC 23 sequences were incorporated into the primers used for amplification. The amplified product was cloned into pSB1C3 and designated as BBa_K1418000. Primers were then designed and PCR was performed to remove the stop codon from TaCHL. This amplified product was cloned into pSB1C3 and is designated as BBa_K1418001. By removing the stop codon, in frame fusions on the 3' end of the gene can be performed. Part BBa_K1418001 was then cloned behind a lac inducible promoter and a ribosome binding site (K208010 contains both R0010 and B0034). To aid in protein purification, a 10x histidine tag with two transcriptional terminators was cloned in frame on the 3' end to generate the final composite construct, BBa_K1418002.

Chlorophyllase Purification

To test for protein production from the new BBa_K1418002 construct, protein purification using a nickel column was performed. Using methods provided in our "protocols" section, cells containing BBa_K1418002 were grown overnight, pelleted and lysed. After centrifugation, the supernatant was applied to the nickel column. Various samples throughout the purification process were analyzed using SDS-PAGE. The SDS-PAGE below shows results from analysis of cells containing BBa_K1418002.

It can clearly be seen from the SDS-PAGE that a protein product between 25kDa and 37kDa has been purified using the nickel column. Since the expected size of our chlorophyllase construct is 34 kDa, we are confident that we have purified the chlorophyllase protein!

Chlorophyll Extraction From Spinach Leaves

Chlorophyll was extracted from spinach leaves using a modified version of the one found in Arkus et. al., 2008. Six spinach leaves were ground with a mortar and pestle. Fifteen ml of chloroform:methanol (1:2 v/v) was added to the mixture and incubated at room temperature for 30 minutes. The solution was then passed through a cheesecloth into a glass beaker. Three ml’s of the solution was transferred to another glass bottle and 1 ml of chloroform with 1.8 ml of ddH2O were added. The vial was shaken to mix and centrifuged to separate phases. The upper aqueous phase was discarded and the lower organic phase was transferred to a new tube. Two to three drops of ethanol were added to clarify solution and allowed to dry out in fume hood overnight. The chlorophyll was resolubilized in 1 ml of acetone. Concentration of chlorophyll was found using a UV-vis spectrophotometer at a 1:100 dilution. Absorbances were read at 663 nm and 646 nm. The concentration of chlorophyll in the dilution equals 0.01776 X A646nm + 0.00734 X A663nm. Molarity was calculated using the molecular weight of chlorophyll, 839.5 g mol-1. The stock was stored in a -20 oC freezer.

We extracted 43.14 ± 8.93 uM of chlorophyll from six spinach leaves that was later used to test the activity of our produced chlorophyllase.

Activity Assay

Chlorophyllase was tested using a chlorophyllase activity assay. Chlorophyll A in acetone, 0.14 mM, was mixed with MOPSO buffer, 20% v/v. Purified protein, 0.1mg, was added in a 1.0 ml reaction volume at 25 C and shaken by hand for 10 min. The reaction was quenched with a 4:6:1 (v/v/v) acetone:heptane:KOH (1 ml). After mixing the reaction tubes were centrifuged for 10 min. and 2,500g to separate phases. The aqueous phase containing the chlorophyllide reaction product was recovered and absorbance measured at 667 nm using a UV-vis spectrophotometer. Using an extinction coefficient of 76.79 mM-1 cm-1 was used to determine chlorophyllide concentrations (Arkus et. al., 2008).

The results showed an increase of 3.527 uM in concentration of chlorophyllide over the control. If the reaction runs well enough, a visual may be seen of the green color changing locations from the organic phase to the aqueous phase, after centrifugation, because of the cleaving of the phytol tail. The photo below shows two layers in each tube, the top organic layer and the bottom aqueous layer. Because the phytol tail makes the chlorophyll molecule nonpolar, it resides in the upper organic layer. Once the chlorophyllase cleaves this tail, it becomes soluble in the aqueous phase and then resides in the lower phase of the tubes shown. Left tube shows before addition of chlorophyllase enzyme and the right tube is after cleavage of the phytol tail has occurred.

References

Tymoczko, J., Ber. J., Stryer, L. (2010) Biochemistry a short course. Ahr, K. (Ed.). New York, NY: W.H. Freeman and Company

Rodriguez, M (2003). Difficulty of grass stain removal. Message posted to http://www.newton.dep.anl.gov/ askasci/gen01/gen01433.htm

Arkus, K. A., Jez, J. M. (2008). An integrated protein chemistry laboratory. Biochemistry and Molecular Biology Education, 36(2), 125-128.

Eckhardt, U., Grimm, B., & Hörtensteiner, S. (2004). Recent advances in chlorophyll biosynthesis and breakdown in higher plants. Plant molecular biology, 56(1), 1-14.

Tymoczko, J., Ber. J., Stryer, L. (2010) Biochemistry a short course. Ahr, K. (Ed.). New York, NY: W.H. Freeman and Company

Rodriguez, M (2003). Difficulty of grass stain removal. Message posted to http://www.newton.dep.anl.gov/askasci/gen01/gen01433.htm

Arkus, K. A., Jez, J. M. (2008). An integrated protein chemistry laboratory.Biochemistry and Molecular Biology Education, 36(2), 125-128.

Eckhardt, U., Grimm, B., & Hörtensteiner, S. (2004). Recent advances in chlorophyll biosynthesis and breakdown in higher plants. Plant molecular biology, 56(1), 1-14.