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Revision as of 08:55, 16 September 2015

IGEM Bordeaux 2015

Project Results

CHOICE OF MATERIALS

crdS, crdA and crdC genes

✵ crdS gene codes the Curdlan synthase.
✵ crdA gene codes a protein which assists translocation of nascent polymer across cytoplasmic membrane.
✵ crdC gene codes a protein which assists translocation of nascent polymer across the periplasm.
crdA, crdC, crdS genes occupy a contiguous 4,948-bp region in Agrobacterium sp. ATCC31749.

N.B. We tried to work on these three genes. However, amplification attempts by PCR were unsuccessful for crdA and crdC genes. So, in a first time, we focused on cloning crdS gene only.

OsmY promoter

In Agrobacterium sp.ATCC31749, Curdlan production is started after a nitrogen starvation in stationary phase. So we decided to use OsmY promoter (BBa_J45992) characterized by MIT 2006 iGEM team which is active in stationary phase and under high osmotic pressure condition. This promoter imitates Curdlan biosynthesis in E.coli without the nitrogen stress.

Figure 1: Growth dependent regulation with three promoters
(Property of MIT 2006 iGEM team)


M63 and LB media

Curdlan production was carried out in two different media: LB medium and M63 medium.
✵ We worked on M63 medium because a mineral salt medium is used on the literature [1]. M63 is a minimal, low osmolarity medium for E.coli, resulting in slower growth rate of these cells.
→ With this medium of known composition we were able to control parameters for the production of our molecule of interest.
✵We worked also on LB medium because this is the most common medium used in the laboratory.

[1] Dae-Young J, Young-Su C. Improved production of Curdlan with concentrated cells of Agrobacterium sp. Biotechnol. Bioprocess Eng. 2001,6:107-111




Figure 2. Optical Densities were analysed each hour after culture inoculation.
As we can see, the growth is much lower in M63 than in LB medium.
✵ For LB medium, we obtained 0.8 OD after 5h.
✵ For M63 medium, 0.8 OD is obtained much later.
→ So, entire process of production goes on 2 days in LB medium and 3 days in M63 medium.

Figure 2: Bacteria growth in two media

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

The production is done on two steps (in two Erlenmeyer flasks) in order to scale up bacterial biomass and then, to obtain a lot of Curdlan.

In stationary phase, we proceed to a temperature change in order to minimize the persistent bacterial growth, the Curdlan being a secondary metabolite.

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.

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.

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.


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 -0.1902
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


N.B. Tests on plants are in progress. Thanks to M. Anthony Bellee (INRA), We started treatment on grape leaves with both produced and bought Curdlan. We will compare results with untreated leaves.
Tests begin with Curdlan treatment and then with downy mildew infection.

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