Difference between revisions of "Team:Sherbrooke/Results"
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This section shows the reliability of <i>vcrx028</i> as a counter-selection marker.for clean deletion recombineering. For the details on the experiments, please click <a href=Experiments#Cyclic_deletion>here</a>. | This section shows the reliability of <i>vcrx028</i> as a counter-selection marker.for clean deletion recombineering. For the details on the experiments, please click <a href=Experiments#Cyclic_deletion>here</a>. | ||
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<h3>BBa_K1744001 insertion in LacZ and phenotype</h3> | <h3>BBa_K1744001 insertion in LacZ and phenotype</h3> | ||
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This section will discuss BBa_K1744001 functionality as a reporter for recombineering. For details about the experiments leading to these results, please click <a href=Experiments#Cassette_opti>here</a>. | This section will discuss BBa_K1744001 functionality as a reporter for recombineering. For details about the experiments leading to these results, please click <a href=Experiments#Cassette_opti>here</a>. | ||
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Revision as of 00:09, 17 September 2015
Project Results
Overview
Experimentations Results
Project modules
MC96
Project modules
MC96
A thermal experimentation has been the only experimentation done on the MC96 module.
Thermal experimentations results
Has no prototype has been built yet, the only results available are the ones from simulations.
Simulation Results
Some simulation has been done on earlier designs, but none of the final design, due to the complexity of simulating heat pipes. Thus, these results are not finals and will surely improve with the addition of the heat pipes between the Peltier elements and the 96-well aluminium mold.
The following figures represent the repartition of heat at the beginning and the end of a heating speed test:
MC96 Heating speed test image
The final temperature has been achieved in 70 seconds.
The following figures represent the repartition of heat at the beginning and the end of a cooling speed test:
MC96 Cooling speed test image
The final temperature has been achieved in 135 seconds.
Back to MC96 Back to Experimentations Results Back to topMC1.5
Thermal and magnetisation experimentations have been conduct to validate the design of the MC1.5 module. These are the results of those experimentations.
Thermal experimentation results
Simulation Results
These are the simulation results for the latest design of the MC1.5.
The following figures represent the repartition of heat at the beginning and the end of a heating speed test:
MC1.5 Heating speed simulation image
The final temperature has been achieved in 70 seconds.
The following figures represent the repartition of heat at the beginning and the end of a cooling speed test:
MC1.5 Cooling speed simulation image
The final temperature has been achieved in 135 seconds.
Back to MC1.5Thermal Trials Results
- Maintaining a temperature below room temperature test results
- Maintaining a temperature over room temperature test results
- Cooling speed test results
- Heating speed test results
Maintaining a temperature below room temperature test results
These are the results obtained by following this protocol. This table illustrates the relation between the voltages applied to the Peltier element and the set temperature of the aluminium mold.
MC1.5 Calibration table cooling
Voltage (V) | Aluminium mold temperature (℃) |
---|---|
0 | 21.8 |
1 | 18.6 |
2 | 15 |
3 | 12 |
4 | 9 |
5 | 6.5 |
6 | 3.3 |
7 | 1.3 |
- The MC1.5 can reach the low temperature specification of 4℃
- The MC1.5 can reach the temperature stability specification of ±1.5℃
Maintaining a temperature over room temperature test results
These are the results obtained by following this protocol. This table illustrates the relation between the voltages applied to the Peltier element and the set temperature of the aluminium mold.
MC1.5 Calibration table heating
Voltage (V) | Aluminium mold temperature (℃) |
---|---|
0 | 21.8 |
1 | 24.7 |
2 | 28.6 |
3 | 34.5 |
4 | 38.9 |
5 | 43.2 |
6 | 47.9 |
7 | 52.3 |
8 | 58.4 |
9 | 63 |
10 | 68.8 |
11 | 83.3 |
- The MC1.5 can reach the high temperature specification of 80℃
- The MC1.5 can reach the temperature stability specification of ±1.5℃
Cooling speed test results
These are the results obtained by following this protocol. This figure shows the aluminium mold’s temperature versus time for an applied voltage of 15V.
![](https://static.igem.org/mediawiki/2015/7/75/Sherbrooke_MC1.5_Cooling_Speed_Results_15V.png)
MC1.5 cooling speed results for 15V
![](https://static.igem.org/mediawiki/2015/b/b6/Sherbrooke_MC1.5_Cooling_Speed_Results_15.5V.png)
MC1.5 cooling speed results for 15.5V
![](https://static.igem.org/mediawiki/2015/a/ad/Sherbrooke_MC1.5_Cooling_Speed_Results_16V.png)
MC1.5 cooling speed results for 16V
- 15.5V is the optimal voltage to apply to obtain the highest cooling speed
- The MC1.5 can reach the specification of a cooling speed of 0.5℃/s
Heating speed test results
These are the results obtained by following this protocol. This figure shows the aluminium mold’s temperature versus time for an applied voltage of 24V.
![](https://static.igem.org/mediawiki/2015/2/2f/Sherbrooke_Heating_Speed_Results_24V.png)
MC1.5 heating speed results for 24V
- The MC1.5 can reach the specification of a heating speed of 1℃/s
Magnetisation experimentation results
Applying an electromagnetic field on the test tube liquid is one of the key functionality of the MC1.5. These are the results of the experimentation done to validate this feature in the MC1.5.
Magnet attraction results
These are the results obtained by following this protocol. These figures show the displacement of the magnetic beads when an electromagnetic field applied to the test tube.
Magnet attraction test results
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Start | After 30 seconds | After 1 minute and 30 seconds |
- The neodymium magnet is enough powerful to attract the magnetic beads within 3 minutes.
TAC
Thermal and turbidity experimentations have been conduct to validate the design of the TAC module.
Thermal experimentation results
Simulation Results
These are the simulation results for the latest design of the TAC.
The following figures represent the repartition of heat at the beginning and the end of a heating speed test:
TAC Heating speed simulation image
The final temperature has been achieved in 70 seconds.
The following figures represent the repartition of heat at the beginning and the end of a cooling speed test:
TAC Cooling speed simulation image
The final temperature has been achieved in 135 seconds.
Back to TACThermal Trials Results
- Maintaining a temperature below room temperature test results
- Maintaining a temperature over room temperature test results
- Cooling speed test results
- Heating speed test results
Maintaining a temperature below room temperature test results
These are the results obtained by following this protocol. This table illustrates the relation between the voltages applied to the Peltier element and the set temperature of the aluminium mold.
TAC Calibration table cooling
Voltage (V) | Aluminium mold temperature (℃) |
---|---|
0 | 21.8 |
1 | 17.4 |
2 | 14.6 |
3 | 12 |
4 | 8.9 |
5 | 6.1 |
6 | 3.2 |
7 | 1.4 |
8 | -0.5 |
9 | -1 |
- The TAC can reach the low temperature specification of 0℃
- The TAC can reach the temperature stability specification of ±1.5℃
Maintaining a temperature over room temperature test results
These are the results obtained by following this protocol. This table illustrates the relation between the voltages applied to the Peltier element and the set temperature of the aluminium mold.
TAC Calibration table heating
Voltage (V) | Aluminium mold temperature (℃) |
---|---|
0 | 21.8 |
1 | 23.9 |
2 | 28.2 |
3 | 33.4 |
4 | 38.8 |
5 | 43.8 |
- The TAC can reach the high temperature specification of 37℃
- The TAC can reach the temperature stability specification of ±1.5℃
Cooling speed test results
These are the results obtained by following this protocol. This figure shows the aluminium mold’s temperature versus time for an applied voltage of 15V.
![](https://static.igem.org/mediawiki/2015/2/2c/Sherbrooke_TAC_Cooling_Speed_Results_15V.png)
TAC cooling speed test 15V
![](https://static.igem.org/mediawiki/2015/1/13/Sherbrooke_TAC_Cooling_Speed_Results_15.5V.png)
TAC cooling speed test 15.5V
![](https://static.igem.org/mediawiki/2015/c/c2/Sherbrooke_TAC_Cooling_Speed_Results_16V.png)
TAC cooling speed test 16V
- 15.5V is the optimal voltage to apply to obtain the highest cooling speed
- The TAC can reach the specification of a cooling speed of 0.3℃/s over room temperature.
- The TAC can reach the specification of a cooling speed of 0.2℃/s below room temperature.
Heating speed test results
These are the results obtained by following this protocol. This figure shows the aluminium mold’s temperature versus time for an applied voltage of 24V.
![](https://static.igem.org/mediawiki/2015/6/6d/Sherbrooke_TAC_Heating_Speed_Results_24V.png)
TAC heating speed test 24V
- The TAC can reach the specification of a heating speed of 1℃/s
Turbidity experimentation results
The purpose of this experiment is to calibrate the turbidity function on the TAC.
Results
These are the results obtained by following this protocol. The following figure is the calibration curve obtained by making a fit on the data obtained.
![](https://static.igem.org/mediawiki/2015/4/47/Sherbrooke_Optical_density_vs_Amplitude_difference.png)
Calibration curve for turbidity function
- The TAC is able to measure turbidity with ±5% of a reference turbidimeter
Biology Results
- Toxin Efficiency Test
- Integrated Toxin Efficiency Test
- pKD3 cassette insertion in pVCR94
- vcrx027 deletion with BBa_K1744000
- Clean deletion of BBa_K1744000
- BBa_K1744001 insertion in LacZ and phenotype
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Toxin Efficiency Test
Here are the data for the killswitch test done with the cells containing the plasmids pBAD30-[mosT;ccdB;mazF;vcrx028], for experiment details, please click here:
pBAD30-mosT
![](https://static.igem.org/mediawiki/2015/f/f6/Toxins_test_pbad30_most.png)
pBAD30-ccdB
![](https://static.igem.org/mediawiki/2015/7/76/Toxins_test_pbad30_ccdb.png)
pBAD30-mazF
![](https://static.igem.org/mediawiki/2015/a/ac/Toxins_test_pbad30_mazf.png)
pBAD30-vcrx028
![](https://static.igem.org/mediawiki/2015/e/e2/Toxins_test_pbad30_vcrx028.png)
Note that the data were normalized with the initial OD[600]. The horizontal axis shows a time lapse and the vertical one the optical density (OD) measured at 600 nm. The optical density serves as an indicator of cell concentration in the medium, the more concentrated the cells are, the higher the OD. Cells were inoculated in LB broth with glucose as a control to show that OD is a good way to follow cell growth. It was also measured with culture in 1% arabinose LB broth to show the effect of toxin induction on the cells. We can see that mosT and vcrx028 shown the best behavior with absence of growth and cell mortality with arabinose (induced killswitch). This shows that cells die more rapidly than they are able to mutate the toxin sequence or gain resistance. It also shows that both mosT and vcrx028 are the most deadly in the tested toxins.
Back to Biology’s Section Menu
Integrated Toxin Efficiency Test
Here are the data for the integrated killswitch test, for experiment details, please click here :
E. coli BW25113-mosT
![](https://static.igem.org/mediawiki/2015/b/b9/Toxins_integrated_test_most_1.png)
E. coli BW25113-vcrx028
![](https://static.igem.org/mediawiki/2015/7/7f/Toxins_integrated_test_vcrx028_1.png)
Note that the data were normalized with the initial OD[600]. The horizontal axis shows a time lapse and the vertical one the optical density (OD) measured at 600 nm. The optical density serves as an indicator of cell concentration in the medium, the more concentrated the cells are, the higher the OD. Cells were inoculated in LB broth with glucose as a control to show that OD is a good way to follow cell growth. It was also measured with culture in 1% arabinose LB broth to show the effect of toxin induction on the cells. We can see that after 10-12 hours, all the tested clones of mosT recombinants and 1 of the vcrx028 ones started growing even if arabinose was present. Therefore, we conclude that vcrx028 is the most fitted counter-selectable marker for our clean deletion system.
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pKD3 cassette insertion in pVCR94
This section shows the results for pKD3’s cassette insertion in pVCR94 and deletion of vcrx028. This was achieved through PCR screening as described in the experiment section.
As it can be seen on the above gel, all of the clone exhibit the correct size band. The only thing that is not good about this result is that there is non-specific amplification in all of the clones. Thing is that we restarted the PCR verification multiple times but could get rid of the non-specific binding. It might be primer off-target. In the other hand, it would be unlikely that the predicted band could be non-specific because we screened with two primer pairs for confirmation. For the first series of amplification, the clones were tested for 5’ end of the insertion, with a primer binding in the 3’ of the cassette. For the right side of the gel, the same clones in the same order were screened for the 3’ end of the insertion with a primer binding in the 5’ of the inserted cassette. Therefore, no product should be visible if the cassette was not inserted at the right place. We kept clone # 3 for further experiments because it was the one with the lowest background of non-specific amplification.
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vcrx027 deletion with BBa_K1744000
This section shows the results of BBa_K1744000 integration in pVCR94, leading to vcrx027 and pKD3 knock-out. For the details on the experiments, please click here.
The above gel shows a representative success rate of BBa_K1744000. The presence of a band in the red rectangle means the clone is good. The clone order is the same on 5’ and 3’ screening. As you can see, the success rate is really high. There is no bad clones in my screening of 5 different colonies. This represent a 100% success rate. This is confirmed by our wild-type control showing no amplification. However, false positives are still possible. I would recommend to screen at least 5 clones to make sure you get at least one good clone. In our case, clone #2 will be used for further experiments.
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Clean deletion of BBa_K1744000
This section shows the reliability of vcrx028 as a counter-selection marker.for clean deletion recombineering. For the details on the experiments, please click here.
The above picture shows the screening effort used to obtain good recombinant. From our experience, it seems all recombinants after clean deletion of BBa_K1744000 are good after screening. You can see that only the control exhibits a different amplicon syze, and that all clones have the syzed band. This means that vcrx028 can be successfully used as a highly efficient killswitch to counter select cells that did not recombine. The success rate of the method is, so far, of 100%.
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BBa_K1744001 insertion in LacZ and phenotype
This section will discuss BBa_K1744001 functionality as a reporter for recombineering. For details about the experiments leading to these results, please click here.
In order to assay the expression of amilCP in single copy, it was inserted through recombineering in lacZ gene in E. coli K-12 substr. BW25113. The result shown above demonstrate that the insertion had a 100% rate of success. In fact, we screened with two primer pairs. The first one (at left) produce a 875 bp amplicon if insertion of the part in the genome is successful but no bands if it failed. The second primer pair (at right) produces a 1.7 kb band if kanR-amilCP was successfully inserted in the genome and a 1.2 kb band if it did not. Using both results, we can conclude that 100% of the clones are positive. The cells were selected on LB kanamycin 50 µg/mL and ampicilin 100 µg/ml (for the selection of the pSIM6 plasmid important for recombineering). The cells were photographied to show the difference between cells with the plasmidic form of amilCP gene and the inserted version of the gene:
In this previous image, you can see the appearance of colonies on LB agar when they carry plasmidic or genomic amilCP-KanR cassette. As you can see, the expression of amilCP is not sufficient to make the colonies blue even faintly. But, this is not as dramatic as it looks, amilCP can still be used in our experiment by exploiting this aspect. The plasmid carrying amilCP can be detected easily because it makes the colonies deep blue. In a recombineering experiment, the longest part if often the screening. There can be a lot of false positive colonies. Some of those can be plasmidic background. But, with amilCP, the plasmidic background will be eliminated rapidly because the colonies will be deep blue instead of white therefore accelerating the screening and eliminating a good proportion of the background. In an automation perspective, this is really important because a robot often manipulate in a more straightforward manner than a real person and false positive colonies could mean restarting all over again.
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Achievements
The project has been done in the context of a end of baccalaureate project that is ending in December 2015. Thus, some specifications that have not been reached for the IGEM competition could be achieved in the following months.
Specification achieved
Specification not achieved yet
Platform:- Movements of the robotic platform must have a 1 mm precision
- Complete platform must be fully open-hardware and detailed at no more than 10000$
- Must be able to use different kinds of pipette tools at the same time
- Must be able to change tools with ease.
- Have a range of opening from 0 mm to 85 mm
- Must grab as small as 1.5 mL tubes
- Must grab as large as 96-well plates
- Rotate at a speed capable of exerting a minimum gravitational force of 6000G
- Must be equipped with security devices such as detection of abnormal vibration or securing the lid after closing
- Suitable for a 96-well plate (common sized plate for biological manipulations
- Control and maintain temperature cycling between 4 to 80℃ ±1.5℃
- Achieve a cooling and heating ramp of 0.5 to 1℃/s
- Apply an electromagnetic field on demand
- Less than 1000$
- Suitable for a test tube of 1.5mL
- Capable of independent control for each unit of three tubes
- Control and maintain temperature cycling between 4 to 80℃ ±1.5℃
- Achieve a cooling and heating ramp of 0.5 to 1℃/s
- Apply an electromagnetic field on demand
- Less than 1000$
- Suitable for a glass tube having a diameter of 25mm, capacity of 50mL
- Independent control for each tube
- Control and maintain temperature cycling between 0 to 37℃ ±1.5℃
- Achieve a heating ramp of 0.08℃/s
- Achieve a cooling ramp of 0.1℃/s above room's temperature
- Achieve a cooling ramp of 0.025℃/s below room's temperature
- Stirring the liquid (Mixing of bacterial cultures)
- Calculate the optical density of the liquid with a precision of ±5% from a reference turbidimeter
- Less than 1000$
Future Plans
The project has been done in the context of end of baccalaureate project that is ending in December 2015. Thus, further development will be done in the following months. This development will be done on the modules that have not been completed and on the optimization of the platform.
After December 2015, new modules are planned. These are the ideas for new modules.
Modules Planned- Camera on the tool holder with autofocus and image analysis capability
- PCR module
- Cell electroporator
- Pump for greater liquid volume (over 1ml)
- Vacuum for spin column-based nucleic acid purification
- Incubator with temperature and C02 control
These modules could be realized by a new team of the University of Sherbrooke.
Some chemists have shown interest in the platform and its modules. Adaptation of those modules for chemistry could also be a possible avenue for further development