Part:BBa_K1962010
Bile Salts Sensing Device
This is a composite part which consists of the bile salt responsive promoter (BBa_K318514) and a combined ribosome binding site / GFP / terminators biobrick (BBa_E0840). The acrRA operon is found in Salmonella enterica strain LT2 and the promoter sequence contains a RamA (BBa_K1962009) binding sequence.
Usage and Biology
First we wanted to characterise the bile salts sensitive promoter acrRA (BBa_K318514). This part was submitted by the 2012 Wisconsin – Madison team, which consists of the acrRA operon found in Salmonella enterica. This system requires a transcription factor known as RamA which binds to the acrRA operon and activated downstream genes. ramA (BBa_K318516) was previously submitted by the Wisconsin – Madison iGEM team in 2012, however, we were unable to obtain this part and so codon optimised the ramA gene for E. coli and had this synthesised by IDT as a gBlock gene fragment and submitted it as a biobrick.
We cloned gfp (BBa_E0840) downstream of the acrRA promoter in order to detect changes in gene expression. RamA was cloned into the pUniprom vector downstream of the constitutive tat promoter. RamA was amplified with a C-terminal HA tag in order to detect expression of the protein.
To test this part, ramA (BBa_K318516), which had been cloned into a pUniprom backbone. this vector was supplied by Professor Tracy Palmer and it contains a constitutive tat promoter for expression. The pSB1C3- PacrRA-gfp and pUniprom-ramA-HA were transformed into E. coli MG1655 cells, and plated onto cml/amp selective media. Colonies from the Lysogeny Broth (LB) transformation agar plate were streaked on MacConkey agar plates and left overnight at 37oC. They were then imaged with a fluorescence microscope to check for GFP expression (Fig 1).
We then conducted a plate reader experiment to better determine whether the presence or absence of bile salts (in this case Sodium Cholate, which is the salt of cholic acids) would make a significant difference to the activation of the promoter and to test whether the promoter would still be active in minimal media. From Fig 2 we can see that the promoter construct (PacrRA-gfp + ramA) is active with and without the addition of sodium cholate however, the activity in its presence is higher. Sodium cholate appears to have an effect.
Figure 3 shows that in the presence of RamA there is increased GFP fluorescence suggesting that the acrRA promoter is being activated.
Figure 1: Microscopy fluorescence imaging for PacrRA-gfp with and without RamA transcription factor on MacConkey agar plates.
Figure 2:96 well plate reader experiment, measuring OD600nm and GFP fluorescence over 20h. pSB1C3-acrRA-gfp transformed with or without pUniprom- ramA. Control consists of both empty pUniprom and empty pSB1C3. 16h overnights were grown at 37oC and then normalized to an OD600nm of 1 with minimal media. Stock of Sodium cholate (in sterile water) was diluted with minimal media to make up to a concentration of 10µg/ml. Showing the difference in GFP fluorescence per unit absorbance when pSB1C3-PacrRA-gfp is grown in the presence or absence of Sodium cholate (10µg/ml)
Figure 3: 96 well plate reader experiment, measuring OD600nm and GFP fluorescence over 20h. pSB1C3-acrRA-gfp transformed with or without pUniprom- ramA. Control consists of both empty pUniprom and empty pSB1C3. 16h overnights were grown at 37oC and then normalized to an OD600nm of 1 with minimal media.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 471
Illegal XhoI site found at 785 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1508
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