Difference between revisions of "Team:Bordeaux/Results"

LABORATORY WORK

1.Cloning

To achieve our Curdlan production by Escherichia coli, it was necessary to integrate our interest gene crdS controlled by the promoter OsmY in two types of plasmids:
✵ in pSB1C3 plasmid for the characterization of our biobricks
• OsmY promoter only
• crdS gene only
• promoter and gene
✵ in pUC for production steps
• promoter and gene

3.Curdlan production

4.Purification and Quantitative analysis

To obtain Curdlan, cells were chemically destroyed by NaOH and many centrifugations.
Curdlan purification was performed by neutralization after adding acetic acid.
Quantitative analysis was done before and after purification to compare and eliminate non significant measures (background signal).

N.B. Also, we use a polarimeter to characterize our Curdlan molecule produced.

2.Transformation

These plasmids are then transferred into competent bacteria E. coli DH5α by transformation. The selection of transformed bacteria is done by chloramphenicol resistance for pSB1C3 plasmid and by ampicillin resistance for pUC plasmid.

To check that cloning work, plasmids are digested with EcoR1 and Pst1 restriction enzymes.

Figure 3:Agarose electrophoresis gel

Figure 3. As we can see, the band corresponding to the piece of linearized plasmid containing OsmY promoter and crdS gene is a bit higher than the band corresponding to the piece of linearized plasmid containing crdS gene only.

E. coli RESULTS

We produced some Curdlan compared to non transformed strain of DH5α. We analyzed data with a student test to prove our results are significant. The theoretical concentration obtained in the Control condition corresponds to the non significant measure (background signal).
N.B. We realized these figures by doing the average of all production results in each medium.

Figure 5: Quantitative analysis of purified Curdlan with aniline blue

→ 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 have studied production in M63 and LB media started at the same time.

Figure 6: Quantitative analysis of purified Curdlan with aniline blue

→ Now, thanks to a statistic test, we can conclude that Curdlan production is doubled in LB medium compared to M63 medium.

LB medium has an unknown glucose concentration contained in yeast extract whereas in M63 medium we have controled this parameter. We suppose that LB glucose concentration is higher than in the second medium. That will be explain the difference between these two conditions.

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.
XXX à finir XXX

Characterization

Part 1. To verify OsmY promoter is only active in the stationary phase, we realized Curdlan quantitative analysis every hour of a culture in LB medium. The switch of temperature for the culture is linked to the transition in stationary phase.
→ As we can see, Curdlan appears after the switch at 27°C. So, OsmY promoter is active in stationary phase only. (Fig.7)

Figure 7: OsmY promoter characterization

Part 2. To verify produced molecule is Curdlan, we analysed samples with a polarimeter. The reference used was Curdlan bought to do standard range 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	-0.2002
produced Curdlan	-0.1982
Control	XXX

At the end...

We have obtained 3.44g of Curdlan in one month of production and with one single gene!

photo à mettre

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