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.: from the same bunch of bananas).

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.