Team:UNITN-Trento/Project/Fruit ripening

From 2013.igem.org

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                                 Note:
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                                 Too high temperature, lycopene synthesis inhibited
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                                 Lycopene synthesis inhibited by the high temperature in the lab
                                  
                                  
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                 In summary: cell transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> (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 decreased activity of lycopene synthesis enzymes. [reference]
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                 In summary: cell transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> (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]
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             <span class="sub-subtitle">&raquo; Iodine Coloration Test</span>
             <span class="sub-subtitle">&raquo; Iodine Coloration Test</span>
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                 Starch accumulates in apples during growing season and is hydrolyzed to sugar in the latter stages of maturation and development (Krotkov and Helson 1946). Hydrolysis occurs in the core area first 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. By relating the pattern of color development to the stage of maturation. <br/>In addition we are performing the same test on bananas.
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                 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. <br/>
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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 to kiwifruit and bananas.
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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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                 <img src="http://2013.igem.org/wiki/images/e/ec/Tn-2013-fruit-Kiwi%27s_compressive_strength.png" style="max-width:800px; box-shadow: 2px 2px 4px #323232;" />
                 <img src="http://2013.igem.org/wiki/images/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> For each sample we tested 5 cube-standard size. In graph one of each sample and the relative ultimate strength. We can show the great difference between the ripened kiwi and the unripened (control and not induced).</span>
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                 <span class="caption justify"><b>Figure 1:</b> After exposure to ethylene produced by BBa_K651001 for XX days the fruit were cut into small cubes of the same size. In graph one of each sample and the relative ultimate strength. For each fruit we tested 5 samples. Negative controls were treated and analysed with the same method. Kiwifruit exposed to ethylene were significantly more ripen than the two negative controls used (no cells and non induced cells transofrmed with Bba_k1065001).</span>
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Revision as of 22:05, 18 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, 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.

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)

Cherry plums are successfully ripened

Type of fruit Cherry plum

Duration of experiment 4 days

Exposition to Ethylene 4 days

Container Fruit in 1 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

Heirloom tomatoes are successfully ripened

Type of fruit Heirloom tomatoes

Duration of experiment 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

Date tomatoes aren't successfully ripened

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 inhibited by the high temperature in the lab

Blackberries aren't successfully ripened

Type of fruit Blackberries

Duration of experiment 7 days

Exposition to Ethylene 1 day

Container Fruit in 0.5 L jar

Note: Same color as control but greater perfume in the induced blackberries

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 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.

Figure 1: After exposure to ethylene produced by BBa_K651001 for XX days the fruit were cut into small cubes of the same size. In graph one of each sample and the relative ultimate strength. For each fruit we tested 5 samples. Negative controls were treated and analysed with the same method. Kiwifruit exposed to ethylene were significantly more ripen than the two negative controls used (no cells and non induced cells transofrmed with Bba_k1065001).

Figure 2: We measure the average of ultimate strength (for each sample) with all 5 cube-standard size and with the best 3 (less sample). We can show the great difference between the ripened kiwi and the unripened with both the 5 cube-standard size and the 3 cube-standard size.

Figure 3: For each sample we tested 5 cube-standard size. In graph one of each sample and the relative ultimate strength. In this experiment we can show how the ultimate strength of EFE sample is a little bit less than the control. This is due at the treatment that the banana's undergo before the transport.

Figure 4: We measure the average of ultimate strength (for the control sample) with all 5 cube-standard size and with the best 3 (less sample). The other measure are the average of ultimate strength with all 5 cube-standard size. We can show the less difference between the ripened banana's and the unripened.

» Fruit color measurement test

Color area L*a*b* (CIELAB) 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).
We used this test to quantify the difference between the color of ripe and unripe fruit's pulp.

Figure 5: L* represent the luminosity of the pulp's kiwifruit. We can show that the EFE sample is more dark than the control samples.

Figure 6: 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.

Methyl salicylate

Click on the images to view them enlarged.

Date tomatoes are successfully unripened

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 are successfully unripened

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.

Blackberries are ripened

Type of fruit Blackberries

Duration of experiment 7 days

Exposition to MeSA (BBa_K1065106) 1 day

Container Fruit in 0.5 L jar

Note: Same color as control. The fruit was in a too greater stage of maturation to block the ripening.

Cherry 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.

Cavendish bananas are ripened

Type of fruit Cavendish banana

Duration of experiment 6 days

Exposition to pure MeSA (LB-dissolved) 6 days

Container Essicator (10.3L)

Note: Pure MeSA dissolved in LB probably don't work

Cherry plums are ripened

Type of fruit Cherry plum

Duration of experiment 4 days

Exposition to pure MeSA (LB-dissolved) 4 days

Container Fruit in 1 L jar

Note: Pure MeSA dissolved in LB don't work, we confirmed that.

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.