Difference between revisions of "Part:BBa K1686046:Experience"
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===Applications of BBa_K1686046=== | ===Applications of BBa_K1686046=== | ||
<H3>'''Part 1 : Curdlan production </H3>''' | <H3>'''Part 1 : Curdlan production </H3>''' | ||
| − | <p align="justify"> We compared the production of Curdlan between a non transformed strain of <i>E. coli DH5α</i> and between our transformed strain with our biobrick. Curdlan was produced in M63 and LB medium at 37°C | + | <p align="justify"> We compared the production of Curdlan between a non transformed strain of <i>E. coli DH5α</i> and between our transformed strain with our biobrick. Curdlan was produced in M63 and LB medium at 37°C. Curdlan production was calculated using aniline blue staning which is specific of beta 1,3 Glucans. Absorption measures at 398nm were done with an excitation wavelength of 398nm and an emission wavelength of 502nm. |
<br> The theoretical concentration obtained in the control condition corresponds to the non significant measure (background signal). We proved that this was a non specific signal with a polarimeter. | <br> The theoretical concentration obtained in the control condition corresponds to the non significant measure (background signal). We proved that this was a non specific signal with a polarimeter. | ||
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https://static.igem.org/mediawiki/2015/thumb/4/49/BordeauxTeam_Mix_LB_and_M63_resultsV3.png/800px-BordeauxTeam_Mix_LB_and_M63_resultsV3.png | https://static.igem.org/mediawiki/2015/thumb/4/49/BordeauxTeam_Mix_LB_and_M63_resultsV3.png/800px-BordeauxTeam_Mix_LB_and_M63_resultsV3.png | ||
| − | <p class="reference" align ="justify"> <b> Figure 1: Quantitative analysis of purified Curdlan with aniline blue in two different mediums with independent experimentations.</b> 2 different mediums were tested: M63 (minimal medium) and LB (complete medium). Production was done at 37°C | + | <p class="reference" align ="justify"> <b> Figure 1: Quantitative analysis of purified Curdlan with aniline blue in two different mediums with independent experimentations.</b> 2 different mediums were tested: M63 (minimal medium) and LB (complete medium). Production was done at 37°C. The results were obtained by calculating the average production in both mediums with independent experiences. Each medium was tested 5 times. </p> <br> |
<p align="justify"> → Each medium can be used for the Curdlan production. But, we can’t compare these two results and say that produced Curdlan quantity is higher in LB than in M63 medium because productions are not realized at the same time in these cases.</p> | <p align="justify"> → Each medium can be used for the Curdlan production. But, we can’t compare these two results and say that produced Curdlan quantity is higher in LB than in M63 medium because productions are not realized at the same time in these cases.</p> | ||
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https://static.igem.org/mediawiki/2015/2/23/Bordeaux_Team_LB_and_M63_resultsV2.png | https://static.igem.org/mediawiki/2015/2/23/Bordeaux_Team_LB_and_M63_resultsV2.png | ||
| − | <p class="reference" align ="justify"> <b> Figure 2: Quantitative analysis of purified Curdlan with aniline blue in two different mediums with parallel experimentations.</b> 2 different mediums were tested: M63 (minimal medium) and LB (complete medium). Production was done at 37°C | + | <p class="reference" align ="justify"> <b> Figure 2: Quantitative analysis of purified Curdlan with aniline blue in two different mediums with parallel experimentations.</b> 2 different mediums were tested: M63 (minimal medium) and LB (complete medium). Production was done at 37°C. The results were obtained by calculating the average production in both mediums with independent experiences. Each medium was tested 5 times. </p> |
Latest revision as of 18:43, 18 September 2015
Applications of BBa_K1686046
Part 1 : Curdlan production
We compared the production of Curdlan between a non transformed strain of E. coli DH5α and between our transformed strain with our biobrick. Curdlan was produced in M63 and LB medium at 37°C. Curdlan production was calculated using aniline blue staning which is specific of beta 1,3 Glucans. Absorption measures at 398nm were done with an excitation wavelength of 398nm and an emission wavelength of 502nm.
The theoretical concentration obtained in the control condition corresponds to the non significant measure (background signal). We proved that this was a non specific signal with a polarimeter.
Figure 1: Quantitative analysis of purified Curdlan with aniline blue in two different mediums with independent experimentations. 2 different mediums were tested: M63 (minimal medium) and LB (complete medium). Production was done at 37°C. The results were obtained by calculating the average production in both mediums with independent experiences. Each medium was tested 5 times.
→ Each medium can be used for the Curdlan production. But, we can’t compare these two results and say that produced Curdlan quantity is higher in LB than in M63 medium because productions are not realized at the same time in these cases.
In following results, we studied the Curdlan production in M63 and LB media starting at the same time. The culture conditions are the same as mentioned above.
Figure 2: Quantitative analysis of purified Curdlan with aniline blue in two different mediums with parallel experimentations. 2 different mediums were tested: M63 (minimal medium) and LB (complete medium). Production was done at 37°C. The results were obtained by calculating the average production in both mediums with independent experiences. Each medium was tested 5 times.
→ We can conclude that Curdlan production is doubled in LB medium compared to M63 medium. This can be explained since LB medium has an unknown glucose concentration contained in yeast extract whereas in M63 medium we have controled this parameter. We suppose that glucose concentration is higher in the LB medium. We can also conclude that in both mediums, Curdlan production is significantly higher in the transformed strains.
However, compared to Saccharomyces cerevisiae, our results are very low: about 10 to 20µg/mL for Bacteria to 100µg/mL for Yeast.
We have tried some optimization protocols but without success.
Part 2. Curdlan characterization
To verify that our produced molecule is Curdlan, we analysed samples with a polarimeter. The reference used was Curdlan which we bought. We used this Curdlan for the standard curve used for the quantitative analysis with aniline blue.
A substance is optically active or has a rotatory power when it deflects polarization plane of light from an α angle. This rotatory power is related to the presence of one or more asymmetric carbon within the molecule. All sugars (except dihydroxyacetone) are chiral molecules, so they all have a rotatory power. This property allows the polarimetric determination of sugars in pure solution thanks to the law of Biot.
There are two types of optically active substances:
✵Those which deflect polarization plane of light from an α angle to the right. The measured angle is positive and the substance is in dextrorotatory form.
✵Those which deflect polarization plane of light from an α angle to the left. The measured angle is negative and the substance is in levorotatory form.
Results
– Results were obtained with a Jasco P-2000 polarimeter.
– Measures are done at 25°C.
| Optical rotation monitor | |
|---|---|
| Reference | -0.1902 |
| produced Curdlan | -0.1982 |
| Control | 0.0590 |
At the end...
We have obtained 3.44g of Curdlan in one month of production and with one single gene!
Perspectives
Despite amplification problems of two genes, we performed cloning with the crdA and crdC sequences. Each gene was placed into plasmids possessing resistance to a different antiobiotic:
pUC-OsmY-crdS (Ampicillin resistance)
pSB1C3-OsmY-crdA (Chloramphenicol resistance)
pSB3T5-OsmY-crdC (Tetracycline resistance)
We have tried without success a triple transformation.
We hope that production yield of Curdlan would be higher if the three genes were present.
| Successful results | Unsuccessful results | Perspectives |
|---|---|---|
| PCR amplification of crdS | No PCR amplification on crdA and crdC | Sulfation step |
| Cloning in pSB1C3 and pUC | No production with transformed bacteria containing the three genes | Test on other E.coli strain |
| Transformation in DH5a strain | Bad purification method | Optimized Curdlan production protocol |
| Curdlan production and characterization | Test of produced Curdlan on plants |
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