Team:UNITN-Trento/Project/Fruit ripening

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             <span class="sub-subtitle">&raquo; Fruit Compression Strength Test</span>
             <span class="sub-subtitle">&raquo; Fruit Compression Strength Test</span>
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               A compression test determines behaviour of materials under crushing loads. The specimen is compressed and deformation at various loads is recorded. Compressive stress and strain are calculated and plotted as a stress-strain diagram, which is used to determine: elastic limit, proportional limit, yield point, yield strength and - for some materials - compressive strength. In our case, we were able to perform a compression strength test with kiwifruit and bananas.
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               A compression test was used to determines maturation stage of kiwifruit and bananas. In this test the fruit is subjected to a cruching loat to measure the resistence of a sample to compression strength. The test allows to measure the strength necessary to break the material analized, as an indication of maturation stage.
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                 <img src="https://static.igem.org/mediawiki/2013/e/ec/Tn-2013-fruit-Kiwi%27s_compressive_strength.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
                 <img src="https://static.igem.org/mediawiki/2013/e/ec/Tn-2013-fruit-Kiwi%27s_compressive_strength.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
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                 <span class="caption justify"><b>Figure 1:</b> Compression test on kiwifruit. After exposure to ethylene produced by BBa_K651001 for XX days the fruit were cut into small cubes of the same size.  For each fruit we tested 5 samples. Negative controls were treated and analysed with the same method. Kiwifruit exposed to ethylene (in green) were significantly more ripen than the two negative controls used (no cells in blue, and non induced cells transofrmed with Bba_K1065001 in red), as demonstrated by the lower strength needed to compress the fruit.</span>
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                 <span class="caption justify"><b>Figure 1:</b> Compression test on kiwifruit. After exposure to ethylene produced by BBa_K651001 for 8 days the fruit were cut into small cubes of the same size.  For each fruit we tested 5 samples. Negative controls were treated and analysed with the same method. Kiwifruit exposed to ethylene (in green) were significantly more ripen than the two negative controls used (no cells in blue, and non induced cells transofrmed with Bba_K1065001 in red), as demonstrated by the lower strength needed to compress the fruit.</span>
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                 <img src="https://static.igem.org/mediawiki/2013/9/9d/Tn-2013-fruit-Comparison_of_ultimate_strength_in_kiwi.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
                 <img src="https://static.igem.org/mediawiki/2013/9/9d/Tn-2013-fruit-Comparison_of_ultimate_strength_in_kiwi.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
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                 <span class="caption justify"><b>Figure 2:</b> Average of ultimate strength for kiwifruit treated with ethylene and negative controls. Kiwifruit exposed to ethylene produced by BBa_K1065001 and negative controls were left for xx days in airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimension from the same fruit. For a comparison we also estimated the strength based the best 3 measurements obtained.  A significant difference between the treated and untreated kiwis is evident, indicating that our ethylene producing device (BBa_K1065001) successfully ripen the kiwis exposed to ethylene after xx days. b.</span>
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                 <span class="caption justify"><b>Figure 2:</b> Average of ultimate strength for kiwifruit treated with ethylene and negative controls. Kiwifruit exposed to ethylene produced by BBa_K1065001 and negative controls were left for 8 days in airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimension from the same fruit. For a comparison we also estimated the strength based the best on 3 measurements obtained.  A significant difference (25 fold less) between the treated and untreated kiwis is evident, indicating that our ethylene producing device (BBa_K1065001) successfully ripen the kiwis exposed to ethylene after 8 days.</span>
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                 <img src="https://static.igem.org/mediawiki/2013/8/80/Tn-2013-fruit-Banana%27s_compressive_strength.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
                 <img src="https://static.igem.org/mediawiki/2013/8/80/Tn-2013-fruit-Banana%27s_compressive_strength.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
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                 <span class="caption justify"><b>Figure 3:</b> Bananas compression test.  After exposure to ethylene produced by BBa_K651001 for XX days the bananas were cut into small cubes of the same size.  For each banana we tested 5 samples. Negative controls were treated and analysed with the same method. Banana exposed to ethylene (in green) were slightly more soft than the two negative controls used (no cells in blue, and non induced cells transofrmed with Bba_K1065001 in red).</span>
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                 <span class="caption justify"><b>Figure 3:</b> Bananas compression test.  After exposure to ethylene produced by BBa_K651001 for 5 days the bananas were cut into small cubes of the same size.  For each banana we tested 5 samples. Negative controls were treated and analysed with the same method. Banana exposed to ethylene (in green) were slightly more soft than the two negative controls used (no cells in blue, and non induced cells transofrmed with Bba_K1065001 in red).</span>
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                 <span class="caption justify"><b>Figure 4:</b>  
                 <span class="caption justify"><b>Figure 4:</b>  
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Average of ultimate strength for bananas treated with ethylene and negative controls. Bananas exposed to ethylene produced by BBa_K1065001 and negative controls were left for xx days in airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimensions from the same fruit. For a comparison we also estimated the strength based the best 3 measurements obtained.  Treated bananas are xx fold less firm than untreated bananas.</span>
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Average of ultimate strength for bananas treated with ethylene and negative controls. Bananas exposed to ethylene produced by BBa_K1065001 and negative controls were left for 5 days in airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimensions from the same fruit. For a comparison we also estimated the strength based the best 3 measurements obtained.  Treated bananas are 1.5 fold less firm than untreated bananas.</span>
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             <span class="sub-subtitle">&raquo; Fruit color measurement test</span>
             <span class="sub-subtitle">&raquo; Fruit color measurement test</span>
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                 Color area L*a*b* (CIELAB)
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                 We used this test to quantify the difference between the color of ripe and unripe fruit's pulp.<br/>
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                 The most used color area is the CIELAB area that uses L* (lightness), a* (redness) and b* (yellowness) parameters. This particular color area is the most complete color space and it describes all the colors visible to the human eye and was created to serve as a device-indipendent model to be used as a reference. [16] The L* parameter indicate a range between yields black (0) and diffuse white (100), the a* parameter indicate a range between red (+a*) and green (-a*, complemental color of red), the b* parameter indicate a range between yellow (+b*) and blue (-b*, a complemental color of yellow). <br/>
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                We estimated the color of kiwifruit using  a high resolution miniature spectrometer, connected to a optic fiber.<br/>
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                We used this test to quantify the difference between the color of ripe and unripe fruit's pulp.
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                 The instrument measures three parameters L* (lightness), a* (redness) and b* (yellowness).  
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                The L* parameter indicate a range between black (0) and white (100), the a* parameter indicate a range between red (+a*) and green (-a*, complemental color of red), the b* parameter indicate a range between yellow (+b*) and blue (-b*, a complemental color of yellow).
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                A shift in the color toward a darker green and a decrease in lightness are an indication of kiwi ripening.
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                 <img src="https://static.igem.org/mediawiki/2013/f/fc/Tn-2013-fruit-Lightness_of_kiwifruit.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
                 <img src="https://static.igem.org/mediawiki/2013/f/fc/Tn-2013-fruit-Lightness_of_kiwifruit.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
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                 <span class="caption justify"><b>Figure 5:</b> L* represent the luminosity of the pulp's kiwifruit. We can show that the EFE sample is more dark than the control samples.</span>
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                 <span class="caption justify"><b>Figure 5:</b> Lightness measurament of kiwifruit treated for 8 days with ethylene (BBa_1065001 induced, blue) and negative controls (No cells, green and BBa_1065001 not induced, red). Treated kiwifuit have a lower L* value as a proof of advanced stage of ripening. </span>
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                 <img src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013-fruit-Color_measurement_of_kiwifruit.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
                 <img src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013-fruit-Color_measurement_of_kiwifruit.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
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                 <span class="caption justify"><b>Figure 6:</b> B* represent the yellow intensity, A* represent the red intensity. This experiment allow to quantify the normal different color between ripened and unripened kiwifruit. That confirm that the our construct that produce ethylene work in order to ripened more quick a fruit.</span>
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                 <span class="caption justify"><b>Figure 6:</b> Color measurement of kiwifruit. Average B* (yellow intensity) and average A* (red intensity) were calculated on 5 slices of the same kiwifruit. Kiwi treated with ethylene (BBa_1065001 induced, blue) show a consistent shift toward a darker green shade respect to controls (no cells, green and BBa_1065001 not induced, red).</span>
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             <p>
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                 Cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> (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 GCMS-FID 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.
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                 Cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> (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 GCMS-FID that 1 ml (analized GCMS-FID) of bacteria culture (induced at O.D.600 equal to 0.6) transformed with our device produce successfully MeSA at a concentration of 0.4mM 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.
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                 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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                 Exposure to pure MeSA coused a toxic effect on cherry tomatoes. We also tested the effect of pure MeSA dissolved in LB but we obtain inconsistent results. For example in a few cases we observed an unexpected change color in the skin of plums and banana.
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                MeSA was reported infact to slow down ripening at high concentration (0.5mM) and to accelerate the process at lower concentration (0.01mM). [The dual effects of methyl salicylate on ripening and expression of ethylene biosynthetic genes in tomato fruit. Ding, C. and Wang, Y. 164, 2003, Plant Science, pp. 589-596.] We think that under the used conditions its concentration was above the threshold required to inhibit ripening.
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Revision as of 11:23, 19 September 2013

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, respectively.

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).

The results obtained varied according to the fruit tested. We tested: bananas, plums, cherries and heirloom tomatoes, blackberries, kiwifruit and others. Results were evaluated qualitatively by observing skin color changes and pulp texture or firmness.

In some cases we were able to detect different levels of fragrance, however this results were too subjective and we discarded them.

We were also able to perform a iodine coloration test on starch-containing fruit.

We also performed quantitative evaluations of ethylene effects by fruit compression strength test.

Ethylene

Click on the images to view them enlarged.

Cavendish bananas are successfully ripened

Type of fruit Cavendish banana

Duration of experiment 6 days

Exposition to Ethylene 6 days

Container Essicator (10.3L)

Ripen plums become red

Type of fruit Plums

Duration of experiment 4 days

Exposition to Ethylene 4 days

Container Fruit in 1 L jar

Heirloom tomatoes ready to be eaten

Type of fruit Heirloom tomatoes

Duration of experiment 9 days

Exposition to Ethylene 9 days

Container Fruit in 1 L jar

Cherry tomatoes are successfully ripened

Type of fruit Cherry tomatoes

Duration of experiment 8 days

Exposition to Ethylene 1 days

Container Fruit in 0.5 L jar

Cherry tomatoes are successfully ripened

Type of fruit Cherry tomatoes

Duration of experiment 7 days

Exposition to Ethylene 1 day

Container Fruit in 0.5 L jar

"Fried Green Tomatoes"

Type of fruit Date tomatoes

Duration of experiment 7 days

Exposition to Ethylene 2 day w/, 3 days w/o, 2 days w/

Container Fruit in 0.5 L jar

Note: Lycopene synthesis probably inhibited by the high temperature (>30 degrees) in the lab as suggested in the literature

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 experiment did not work because the temperature in the laboratory (in those days) was over 30 degrees, causing a decreased activity of lycopene synthesis enzymes. [reference]

» Iodine Coloration Test

Starch accumulates in apples during growing season and is hydrolyzed to sugar in the last stages of maturation and development (Krotkov and Helson 1946). Hydrolysis occurs first in the core area and progresses outwards (Phillips and Poapst 1952). Starch in cut sections of the fruit reacts with a solution of iodine-potassium iodide to produce a blue-black color. The intensity and pattern of the color developed is an indication of the stage of maturation.

» Fruit Compression Strength Test

A compression test was used to determines maturation stage of kiwifruit and bananas. In this test the fruit is subjected to a cruching loat to measure the resistence of a sample to compression strength. The test allows to measure the strength necessary to break the material analized, as an indication of maturation stage.

Figure 1: Compression test on kiwifruit. After exposure to ethylene produced by BBa_K651001 for 8 days the fruit were cut into small cubes of the same size. For each fruit we tested 5 samples. Negative controls were treated and analysed with the same method. Kiwifruit exposed to ethylene (in green) were significantly more ripen than the two negative controls used (no cells in blue, and non induced cells transofrmed with Bba_K1065001 in red), as demonstrated by the lower strength needed to compress the fruit.

Figure 2: Average of ultimate strength for kiwifruit treated with ethylene and negative controls. Kiwifruit exposed to ethylene produced by BBa_K1065001 and negative controls were left for 8 days in airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimension from the same fruit. For a comparison we also estimated the strength based the best on 3 measurements obtained. A significant difference (25 fold less) between the treated and untreated kiwis is evident, indicating that our ethylene producing device (BBa_K1065001) successfully ripen the kiwis exposed to ethylene after 8 days.

Figure 3: Bananas compression test. After exposure to ethylene produced by BBa_K651001 for 5 days the bananas were cut into small cubes of the same size. For each banana we tested 5 samples. Negative controls were treated and analysed with the same method. Banana exposed to ethylene (in green) were slightly more soft than the two negative controls used (no cells in blue, and non induced cells transofrmed with Bba_K1065001 in red).

Figure 4: Average of ultimate strength for bananas treated with ethylene and negative controls. Bananas exposed to ethylene produced by BBa_K1065001 and negative controls were left for 5 days in airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimensions from the same fruit. For a comparison we also estimated the strength based the best 3 measurements obtained. Treated bananas are 1.5 fold less firm than untreated bananas.

The test helped us to quantify the stage of maturation of kiwifruit and bananas, as another confirmation that our ethylene producing device works as predicted. However the test worked better on kiwis rather than bananas probably because the bananas used started already from a more advanced maturation stage. » Fruit color measurement test

We used this test to quantify the difference between the color of ripe and unripe fruit's pulp.
We estimated the color of kiwifruit using a high resolution miniature spectrometer, connected to a optic fiber.
The instrument measures three parameters L* (lightness), a* (redness) and b* (yellowness). The L* parameter indicate a range between black (0) and white (100), the a* parameter indicate a range between red (+a*) and green (-a*, complemental color of red), the b* parameter indicate a range between yellow (+b*) and blue (-b*, a complemental color of yellow). A shift in the color toward a darker green and a decrease in lightness are an indication of kiwi ripening.

Figure 5: Lightness measurament of kiwifruit treated for 8 days with ethylene (BBa_1065001 induced, blue) and negative controls (No cells, green and BBa_1065001 not induced, red). Treated kiwifuit have a lower L* value as a proof of advanced stage of ripening.

Figure 6: Color measurement of kiwifruit. Average B* (yellow intensity) and average A* (red intensity) were calculated on 5 slices of the same kiwifruit. Kiwi treated with ethylene (BBa_1065001 induced, blue) show a consistent shift toward a darker green shade respect to controls (no cells, green and BBa_1065001 not induced, red).

Methyl salicylate

Click on the images to view them enlarged.

Date tomatoes stay green

Type of fruit Date tomatoes

Duration of experiment 7 days

Exposition to MeSA (BBa_K1065106) 2 day w/, 3 days w/o, 2 days w/

Container Fruit in 0.5 L jar

Cherry tomatoes unripen

Type of fruit Cherry tomatoes

Duration of experiment 7 days

Exposition to MeSA (BBa_K1065106) 7 days

Container Fruit in 0.5 L jar

Note: One of the control was probably at a different maturation state ad day1.

Pure MeSA has a toxic effect on cerry tomatoes

Type of fruit Cherry tomatoes

Duration of experiment 8 days

Immersion in MeSA pure 8 days

Container Fruit in 0.5 L jar

Note: The immersion in MeSA pure killed the fruit.

Cells transformed with 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 GCMS-FID that 1 ml (analized GCMS-FID) of bacteria culture (induced at O.D.600 equal to 0.6) transformed with our device produce successfully MeSA at a concentration of 0.4mM 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 coused a toxic effect on cherry tomatoes. We also tested the effect of pure MeSA dissolved in LB but we obtain inconsistent results. For example in a few cases we observed an unexpected change color in the skin of plums and banana. MeSA was reported infact to slow down ripening at high concentration (0.5mM) and to accelerate the process at lower concentration (0.01mM). [The dual effects of methyl salicylate on ripening and expression of ethylene biosynthetic genes in tomato fruit. Ding, C. and Wang, Y. 164, 2003, Plant Science, pp. 589-596.] We think that under the used conditions its concentration was above the threshold required to inhibit ripening.

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