Team:Valencia Biocampus/Bacterium

From 2012.igem.org

Bacteria Subteam

Here is an overview of how our bacteria work. For more information look the molecular mechanisms below.

MOLECULAR MECHANISMS

LACTOSE-INDUCED PROMOTER

This construction is made up of three parts: (1) the transcription factor-binding site insidethe promoter, (2) the repressor-binding site outside the promoter and (3) the coding sequence, which contains a synthetic fluorescent (blue) protein. Our construction uses the well-known lactose operon system{1}. Since there is an operator region that blocks transcription, it is necessary to know it and avoid it.

When is the protein synthesized? In order to obtain the blue fluorescent protein two conditions have to be met. First condition: there is no glucose in the medium. Second condition: lactose is present in the medium (it also works with other inductors, like IPTG).

The molecular mechanism underlying this phenomenon is as follows: a lack of glucose promotes the formation of CAP (or CRP{2}), which binds to specific sites upstream of sugar-metabolizing genes and activates its transcription. The binding of this molecule depends on the presence of the allosteric effector cAMP (the concentration of this metabolite changes in response to the presence or absence of different nutrients).Moreover, another condition is needed since LacI repressor, produced constitutively by the lacI gene inside the lactose operon, will bind to the operator region and will block the transcription unless lactose is also present in the medium.Once lactose enters the cell it is converted to allolactose{3}, and this molecule binds tightly to the repressor so it can no longer block the transcription. Then, the fluorescent protein can glow in the cytoplasm!

¿How did we deal with this construction?In our experiments we not only tested the differential expression when glucose is absent + lactose present and vice versa, but we also tested the expression and growth rates when different compounds were added as carbon-enriched sources. For example, we added sodium acetate and galactose as substitutes of glucose. We determined the best IPTG concentration for our cultures too.

References:
1. F. Jacob and J. Monod. (1959) Genes of structure and genes of regulation in the biosynthesis of proteins. C. R. Hebd. Seances Acad. Sci.249: 1282–4.
2. S., Busby and R.H., Ebright. (2001). Transcription activation by catabolite activator protein (CAP). J. Mol. Biol.293: 199–213.
3. RE., Huber, K., Wallenfels and G.,Kurz. (1975)The action of beta-galactosidase (Escherichia coli) on allolactose.Canadian Journal of Biochemistry, 53(9):1035-1038


*For lactose-activation, switch glucose for IPTG.

Glucose concentration in lactose-activation media (from a stock of 50 g/L made by Alba & Lamberto): 5 g/L, 1 g/L and 0.1 g/L. Since 0.1 g/L is likely to be the most useful concentration, let’s go on later with 0.05 g/L, 0.01 g/L and 0.001 g/L. Remember to add IPTG!

NITROGEN-REGULATED PROMOTER

This construction contains two parts: (1) the transcription factor-binding site inside the promoter and (2) the coding sequence, which contains a synthetic fluorescent (yellow) protein. We chose as the promoter sequence the one of the glnA gene. There is long evidence that this promoter is regulated by nitrogen concentration{1}. Moreover, the promoter is not the canonical one (70) but an alternative one (54){2},{3}.

When is the protein synthesized? In order to obtain the yellow fluorescent protein a condition should to be met. That condition is related to nitrogen-starvation. The less nitrogen there is, the more expression you get!

The molecular mechanism underlying this phenomenon is as follows: in gram-negative bacteria, transcriptional activation in response to some external stimuli (absence of nitrogen,high UV-dosis,forexample) often involves the alternative sigma factor, 54. This factor, alsocalledRpoNor Sigma N, was originally identified as the sigma factor for nitrogen-controlled genes. 54works in conjunction with members of the NtrC (Nitrogenregulatoryprotein C) superfamily of transcriptional activators. In our case, when ammonia levels are low the bacterium undergoes some metabolic changes. Within these changes, there are some related to nitrogen assimilation and processing, so our construction, which responds to low ammonia levels, increases its transcription.

How did we deal with this construction?We developed a nitrogen-gradation tube experiment, which means that we added different (NH4)2SO4 concentrations to each tube, so we can measure a gradation of the protein fluorescence. We used a synthetic medium for this.

References:
1. Reitzer, L.J., Movsas, B. andMagasanik, B. (1989)Activation of glnA transcription by Nitrogen Regulator I (NRI)- phosphate in Escherichia coli: evidence for a long-range physical interaction between NRI-phosphate and RNA polymerase.Journal of bacteriology, 171 (10):5512-5522
2. Wang, L., Guo, Y. and Gralla, J.D. (1999) Regulation of sigma 54-dependent transcription by core promoter sequences: role of - 12 region nucleotides. Journal of bacteriology, 181 (24):7558–7565
3. Barrios, H., Valderrama, B. y Morett, E. (1999) Compilation and analysis of 54-dependent promoter sequences. Nucleics Acids Research, 27 (22):4305-4313.

OXYGEN-REGULATED PROMOTER