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Experimental results

In this section a summary of "Ethanol? Whey not!" project results is reported. For more information you can visit Parts Characterization section.

Ethanol fermentation

Survival of E. coli TOP10 in ethanol

Different concentrations of ethanol have been added to LB and M9 supplemented with glycerol selective media and E. coli TOP10 (bearing BBa_B0030 BioBrick) survival, when grown in a microplate reader (37°C, automatic protocol) and in a 50 ml falcon tube (37°C, 220 rpm), has been tested.

Of the concentrations we tested, in the microplate reader bacteria can grow at concentrations up to 2.5%w/v (LB) and 1.5%w/v (M9), while in the 50 ml falcon tube they can grow at concentrations up to 3.5%w/v (LB) and 2.5%w/v (M9).

In general, in the falcon tube bacteria grow better than in the microplate (see Measurement section for details) and this is confirmed by these data: it seems that the threshold concentration of ethanol is higher for growth in falcon tube than in the microplate (for example, at 2.5%w/v in M9, bacteria can survive in the falcon tube, but not in the microplate).

Ethanol production

A synthetic ethanol-producing operon () has been built up by our team and has been assembled downstream of several promoters or devices:

• - tetR promoter (in high copy plasmid)
• - constitutive promoter (in high copy plasmid)
• - constitutive promoter (in high copy plasmid)
• - 3OC6-HSL inducible system (in high and low copy plasmids)

These strain's most important phenotype qualitative characteristics can be summarized as follows:

• strains that had tetR promoter, J23106 or J23118 upstream of the operon gave very small colonies when plated on selective LB agar[[Image: plates (with or without 2% of glucose);
• strains with 3OC6-HSL inducible system upetream of the operon in low copy plasmid could grow when induced with 100 nM and 1 uM HSL;
• strains with 3OC6-HSL inducible system upstream of the operon in high copy plasmid died when induced with 100 nM and 1 uM HSL, but could grow when not induced;
• bacterial density of strains with 3OC6-HSL inducible system upstream of the operon in liquid cultures supplemented]] with 2% or 10% of glucose were higher than the density of all other tested strains grown in the same conditions;

All the other details can be found on Parts Characterization page, as well as in Registry page.

These strain's most important phenotype quantitative characteristics can be summarized as follows:

Growth of strains bearing:

• 3OC6-HSL inducible system upstream of the operon in high copy plasmid () uninduced;
• with 3OC6-HSL inducible system upstream of the operon in low copy plasmid () uninduced;
• 3OC6-HSL inducible system upstream of the operon in low copy plasmid () induced with 1 uM of 3OC6-HSL;
• 3OC6-HSL inducible system without the operon in high copy plasmid ()
• a promoterless operon in high copy plasmid ()

has been monitored in selective LB + 10% glucose starting from an inoculum in the microplate reader (figure below).

Ethanol fermentation capability of these strains in 10%w/v glucose-supplemented LB medium has been tested with three different fermentation protocols (see [experiments| Fermentation experiments] and [- ethanol producing device| BBa_K173003 - ethanol producing device] for a detailed description).

The main fermentation results can be summarized as follows:

Lactose metabolism

A bacterial beta-galactosidase protein generator () was assembled from the parts contained in the Registry, even if it had already been designed and submitted as , whose sequence was consistent according to iGEM QC.

The enzyme, encoded by lacZ gene, cleaves lactose in glucose and galactose and can be used, properly assembled downstream of a regulatory module, to speed up the lactose metabolism.

We used tetR promoter, contained in , to produce a constitutive expression of lacZ.

The correct behaviour of the expressed beta-gal enzyme has been tested in TOP10 E. coli (which do not have a working lacZ gene in its genome) on X-Gal plates (more details Parts Characterization section):

Constitutively expressed beta-gal ( in pSB1AK3)	Negative control: TOP10 bearing in pSB1A2
strain, which contains a working lacZ gene	Promoterless beta-gal protein generator ( in pSB1AK3)

This qualitative result shows that:

• beta-gal is correctly expressed in our engineered strain (1st picture), while in E. coli TOP10 (2nd picture) it is not;
• Surprisingly, as reported in the 4th picture, the promoterless protein generator shows blue colour. It may be due to i) spurious transcription of the protein generator in the high copy number plasmid pSB1AK3 or to ii) the recombination occurred between plasmidic lacZ and genomic lacZdeltaM15, in which the working lacZ was integrated in E. coli genome under the control of lac promoter. This phenomenon has still to be studied. Caltech iGEM 2008 team reported this phenomenon in a similar protein generator, in which beta-gal assay was performed.

Anyway, further comparative tests should be done in order to see if lactose cleavage can be performed faster than in wild type E. coli, after the choice of a suitable promoter which controls this protein generator.

Promoter characterization

Several promoters and devices have been tested by our team during this summer. Our efforts have been focused on standard characterization methodologies, so we decided to try to apply the Relative Promoter Units (Kelly J. et al., 2008) concept to do this. Regulation of gene expression is very important for our project as we want to optimize the expression rate of two actuators, one for lactose conversion to glucose and one for ethanol production from glucose. In our case it essential do have a sort of "genetic regulators user's handbook", in which all the functional characteristics of promoters can be found and in which we can choose the suitable regulator for us.

We dedicated a large part of our project in this direction and here we summarize the promoter strength measurements. In the following bar plot we report:

• X axis - the name and the working condition (medium, induction) of the promoter or device;
• Y axis - the measured strength of the tested promoter or device reported in RPUs, as well as the minimum and the maximum measured values.

File:Pv promoters bar.jpg

Project conclusions and perspectives