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.

••••• Parts Collection 2016

This is part of a Part Collection of 18 BioBricks designed by Dundee iGEM 2016. This collection will be useful to teams working with toxins as we have submitted new toxins to the registry. Working with bacterial toxins is difficult due to the risk of toxicity to the chassis, so the corresponding immunity for our toxins were also submitted. We have also submitted these toxins lacking their cytotoxic domains replacing it with a multiple cloning site which will allow for different toxic domains to be fused at the C-terminus and thereby generating a synthetic toxin. In addition, there are three well-characterised promoters that can be used to initiate gene expression at various points in the digestive tract, to enable devices to function within a human or animal. Finally, a lysis cassette was constructed to lyse or burst cells, thus releasing the toxins and destroying the GM bacteria to prevent its release to the environment. This BBa_K1962010 is a critical member of the Parts Collection very important for measuring promioter activity in response to bile salts. Use in conjunction with BBa_K1962009.

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), was loned into a pUniprom backbone, this vector was supplied by Professor Tracy Palmer and contains a constitutive tat promoter for expression. This was then used in conjunction with the bile salts sensing device. The bile salts sensing device (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 in minimal media to 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 further activated by RamA.

In order to understand if the growth medium plays a role in the activation of the acrA promoter cells harbouring pacrRA-gfp and ramA were grown in LB and cells harbouring only pacrRA-gfp without ramA were also grown. And in Fig 4 you can see that after a anti-GFP blot was carried out, you could see GFP production even in the absence of RamA, it is difficult to tell if there is any difference in the amount of GFP being detected in the presence of RamA.

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.

Figure 4: MG1655 cells harbouring pSB1C3-PacrRA-gfp and pUniprom-ramA wre grown overnight at 37oC. 1 ml of the overnight culture was pelleted and re-suspended in 100 ul Laemmli buffer and 15 ul samples were then separated by SDS PAGE (12% acrylamide) and transferred to PVDF membrane followed by probing with anti-GFP antibody.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 471
  Illegal XhoI site found at 785
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1508