Team:Dundee/Results

Results

Promotors

pH Sensing Device

Introduction

Bacteria have the ability to sense their surroundings and respond to them by turning on genetic regulatory systems. One of the environmental stimuli to which bacteria may respond is pH1. Bacteria respond to environmental stimuli for a variety of reasons for example to trigger pathogenesis or survival genes, which help the bacterium, survive, thrive and compete in a new environment1.

We found several promoters that were submitted from previous iGEM teams, this included Pasr (BBa_K1231000) and PgadA (BBa_K1231001) both submitted by the 2013 Northwestern iGEM team. These have been reported to be activated at low pH. So we started by characterising these two promoters and finding the best one suitable for our project. We cloned gfp (BBa_E0840) downstream of both of these promoters in order to measure expression levels (enter biobrick numbers for our constructs) For our bile salt responsive promoter we used acrRA (BBa_K318514), submitted by the 2012 Wisconsin –Madison team, which consists of the acrRA operon found in salmonella enterica strain. This operon contains the RamA binding sequence, the acrR gene and part of the acrA gene. We then cloned gfp (BBa_E0840) downstream of acrRA (include biobrick number). Due to E. coli not containing its own RamA transcription factor we codon optimised the ramA gene sequence for expression in E. coli and got it synthesised by IDT as a gBlock gene fragment. RamA was also submitted by the Wisconsin-Maddison 2012 iGEM team (BBa_K318516) but was out of stock in the iGEM registry so we also submitted this as a biobrick (enter biobrick number).

Pasr

Enterobacteria can respond to low pH by de novo synthesis of specific proteins and altered levels of gene expression. The response to environmental stresses, such as pH, is often a complex mechanism and also depends on other environmental factors such as nutrition, the presence or absence of oxygen or starvation2. The E. coli asr (acid shock RNA) gene encodes small RNA, of about 450 nucleotides in length2. This gene is inducible by low external pH and contributes to the organism’s survival2. It has been suggested that asr genes may be regulated by the two component system phoB-phoR2.

In this two-component system the protons from the environment (H+) activate the sensory part (phoR- in the periplasm) of the two-component system, which then transduces the signal to the activator protein- phoB, which can bind promoter DNA of asr2. The promoter region of asr was analysed and showed to contain a sequence similar to the pho-box; this is a consensus sequence able to bind to the phoB protein2. These interactions of H+ from the external environment with this two-component system are thought to lead to asr transcription2. Interestingly the phoB-phoR system also controls the pho regulon which is induced by phosphate starvation, the link between these two factors is not fully understood however, it has been suggested that the level of asr expressed in minimal media is higher than in enriched media2.

PgadA

Bacteria have a variety of environmental response mechanisms; the GAD (glutamate decarboxylase) system in E. coli has been suggested to be the most effective response to environmental acidic conditions3. This system uses two main isoforms – gadA and gadB and a putative glutamate/Q-amino butyric acid antiporter encoded by gadC3. By decarboxylation of glutamate the protons that leak into the cell can be consumed4. The end product, γ-aminobutyric acid (GABA), is then transported out of the cell by gadC4. The control of this system is very complex involving two repressors (H-NS and cyclic AMP receptor protein), one activator (gadX), one repressor activator (gadW) and two sigma factors (σS and σ70) 3.

Design

The device was designed to be active upon ingestion at a pH of approx. 5.5 or below in order to release our colicins. The promoter is not constitutive and activates transcription of genes at a low pH. The pH range of the promoter was investigated in this project.

In 2013 the Northwestern iGEM team submitted two pH sensitive promoter Pasr (BBa_K1231000) and PgadA (BBa_K1231001). In order to further characterise both of these promoters we cloned gfp (BBa_E0840) downstream of both promoters (Fig 1). We then used this construct to measure and compare the GFP expression levels in response to different pH conditions.

Figure 1: Schematic representation of pH sensitive promoters-gfp constructs in pSB1C3 plasmid.

We wanted to test both our pH sensitive promoters, Pasr and PgadA by monitoring for the production of GFP by western blotting. The western blots showed levels of GFP protein production by the cells at a range of pH values. In Fig 2A the expression of GFP at pH 5 is much higher than the other pH’s. This would indicate that at this pH the largest amount of GFP was being produced under the induction of the gadA promoter. The promoter is slightly leaky as there is also expression of protein within cells at other pH values. In Fig 2B the GFP protein expression under the regulation of Pasr can be seen, the range of activity for this promoter is much wider indicating that this promoter is leakier than PgadA. The level of expression of GFP under the regulation of Pasr appears to be uniform with only a slight increase in GFP at pH5.

Figure 1: Schematic representation of pH sensitive promoters-gfp constructs in pSB1C3 plasmid.