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Team:UNITN-Trento/Project/Fruit ripening
From 2013.igem.org
Fruit ripening
We have tested several type of fruit to verify that our engineered bacteria are able to produce ethylene (cell transformed with BBa_K1065001) and methyl salicylate (cell transformed with BBa_K1065102 and BBa_K1065106) for enhancing or blocking the ripening of fruit.
The flasks containing the induced culture were kept at 37 degree in agitation and connected to a sealed jar (or a desiccator), where the fruit was exposed to either ethylene or methyl salicylate (MeSA). Negative controls were either a fruit kept in airtight jar or a fruit placed in a jar connected to a flask with non-induced cells.
Within the same experiment we used fruit assumed to be at the same ripening stage (i.e.: the same bunch of bananas).
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Type of fruit Cavendish banana Duration of experiment 6 days Exposition to Ethylene 6 days Exposition to pure MeSA (LB-dissolved) 6 days Container Essicator (10.3L) Comment Maturation started 4 days after exposition to Ethylene. Methyl Salicylate in this experiment seems to ripen the fruit. |
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Type of fruit Cherry plum Duration of experiment 4 days Exposition to Ethylene 4 days Exposition to pure MeSA (LB-dissolved) 4 days Container Fruit in 1 L jar Comment Ethylene successfully ripened plums after 4 days. Methyl salicylate in this experiment seems to ripen the fruit. We performed the experiment in duplicate, in a different jar (1.5L) and we obtained the same results. |
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Type of fruit Cherry tomatoes Duration of experiment 7 days Exposition to Ethylene 1 day Exposition to MeSA (BBa_K1065106) 7 days Container Fruit in 0.5 L jar Comment Ethylene successfully ripened the Cherry tomatoes. MeSA present the same coloration and consistence as the two control. One of the tomatoes in the control ripened, probably due at different stage of maturation of the fruit when it was placed in the jar. |
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Type of fruit Heirloom tomatoes Duration of experiment 9 days Exposition to Ethylene 2 days Container Fruit in 1 L jar Comment Ethylene successfully ripened the tomatoes. |
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Type of fruit Date tomatoes Duration of experiment 7 days Exposition to Ethylene 2 day w/, 3 days w/o, 2 days w/ Exposition to MeSA (BBa_K1065106) 2 day w/, 3 days w/o, 2 days w/ Container Fruit in 0.5 L jar Comment No difference showing compared with the control. Given that the tomatoes exposed at 30°C in our lab, we confirmed that the hot temperature block the ripening of the tomatoes. [reference] |
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Type of fruit Cherry tomatoes Duration of experiment 8 days Exposition to Ethylene 1 days Immersion in MeSA pure 8 days Container Fruit in 0.5 L jar Comment In this experiment the fruit was immersed in pure MeSA and then left in the jar for the remaining time of the test. MeSA had a toxic effect on the fruit, as it can be observed by skin and pulp colour. Ethylene work as aspected. |
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Type of fruit Blackberries Duration of experiment 7 days Exposition to Ethylene 1 day Exposition to MeSA (BBa_K1065106) 1 day Container Fruit in 0.5 L jar Comment All Blackberries ripened simultaneously (colour change), however fruit exposed to ethylene had a more intense and typical smell of blackberries. |
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Type of fruit Cavendish banana Duration of experiment 10 days Exposition to Ethylene 10 days Exposure to pure MeSA 10 days Container Fruit in 1.5 L jar Comment In this experiment the fruit was placed in a jar containing pure MeSA, but was not in direct contact. MeSA had a toxic effect on the fruit, as it can be observed by skin and pulp colour.. |
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Type of fruit Cherry plum Duration of experiment 14 days Exposition to Ethylene 2 day w/, 2 days w/o, 2 days w/ Exposition to MeSA (BBa_K1065106) 2 day w/, 2 days w/o, 2 days w/ Container Fruit in 1.5 L jar Comment Despite the coloring did not give visible results, the text of the starch demonstrates how the apple subjected to ethylene is more ripened compared to the control. MeSA seems in a middle stage between control and ethylene treated. |
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The results obtained varied according to the fruit tested. We tested: bananas, cherry plums, cherries and heirloom tomatoes, plums, blackberries, kiwifruit, pears and others. Results were evaluated qualitatively by observing skin color changes and pulp texture or firmness. We were also able to perform a iodine coloration test on starch-containing fruit.
In some cases we were able to detect different levels of fragrance, however this results were too subjective and we discarded them. We also performed quantitative evaluations of ethylene effects by fruit compression strength test.
In summary: cell transformed with BBa_K1065001 (our ethylene producing device) successfully ripen all the fruit tested when compared to the negative control, except for a few cases (Test with Date Tomatoes) where no significant effects were observed. We believe that in the first case the experiment did not work because the temperature in the laboratory (in those days) was over 30 degrees. We know the importance of temperature on ripening of vine-rape fruit (with external color). In our specific case the too high temperature (>25°C) caused inhibition of ethylene production which is needed for good lycopene synthesis. [reference]
Cells transformed with BBa_K1065102 and BBa_K1065106 (our methyl salicylate producing devices) did not show any difference when compared to the negative control. We were not able to determine if MeSA actually inhibited fruit ripening because the experiments were not long enough to allow ripening of the control. We demonstrated by XX that YY ml of bacteria culture (induced at O.D.600 equal to 0.6) transformed with our device produce successfully MeSA at a concentration of xx ppm in the culture medium. However, the final concentration of the inhibitor in the gas phase in the jar could be lower than the needed concentration. We are now in the process of better estimating these values.
Exposure to pure MeSA instead caused an excessive ripening when added to the culture medium while it had a toxic effect when the fruit was drenched with it. MeSA was reported to slow down ripening at high concentration (XX) and to accelerate the process at lower concentration (YY). [ref] We think that under the used conditions its concentration was above the threshold required to inhibit ripening.
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