Difference between revisions of "Part:BBa K1962009"

Latest revision as of 23:41, 29 October 2016

Transcriptional Activator RamA

RamA is a transcriptional activator that has been used in conjunction with BBa_K1962010 which is a bile salt sensing device, to induce GFP expression by binding the RamA binding site present on the acrRA promoter.

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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_K1962009 is a critical member of the Parts Collection and must be used to ensure the bile salt promoter (for example BBa_K1962010) is functional.

Usage and Biology

RamA (BBa_K318516), was cloned into a pUniprom backbone which was supplied by Professor Tracy Palmer and it contains a constitutive tat promoter for expression.

In order to further understand the role of RamA we first wanted to determine that we were able to detect the expression of RamA-HA. In Fig 1 the RamA transcription factor containing its HA tag was transformed into Rosetta E. coli strain which contains a viral T7 polymerase, activated by IPTG and you can see successful blotting for RamA-HA.

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 2)..

We then conducted a plate reader experiment to better determine whether the presence or absence of RamA 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 3 we can see that in the presence of RamA there is increased GFP fluorescence suggesting that the acrRA promoter is being activated by RamA.

Figure 1: pUniprom-ramA was transformed into Rosetta cells containing a viral T7 polymerase which is activated by IPTG. Anti-HA tag western blot for RamA in pUniprom within the Rosetta cells (DE3). Cells were subcultured from an overnight at 37OC. Once an OD of 0.4 was reached the samples were added to a final concentration of 1mM IPTG. 1ml of this was pelleted and Laemlli buffer added before loading 15 µl samples onto the protein gel. SDS PAGE (12% acrylamide) was run and transferred to PVDF membrane followed by probing with anti-HA antibody.

Figure 2: Microscopy fluorescence imaging for PacrRA-gfp with and without RamA transcription factor on MacConkey agar plates.

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 RamA is a transcriptional activator that has been used in conjunction with <partinfo>BBa_K1962010</partinfo> which is a bile salt sensing device, to induce GFP expression by binding the RamA binding site present on the <i>acrRA</i> promoter.
-<!-- Add more about the biology of this part here
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_K892008 AddReview 5</partinfo>
+<I>Parts Collection 2016</I>
+|width='60%' valign='top'|
+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 <partinfo>BBa_K1962009</partinfo> is a critical member of the Parts Collection and must be used to ensure the bile salt promoter (for example <partinfo>BBa_K1962010</partinfo>) is functional.
+|}
 ===Usage and Biology===
+RamA (BBa_K318516), was cloned into a pUniprom backbone which was supplied by Professor Tracy Palmer and it contains a constitutive tat promoter for expression.
+In order  to further understand the role of RamA we first wanted to determine that we were able to detect the expression of RamA-HA. In Fig 1 the RamA transcription factor containing its HA tag was transformed into Rosetta E. coli strain which contains a viral T7 polymerase, activated by IPTG and you can see successful blotting for RamA-HA.
+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 2)..
+We then conducted a plate reader experiment to better determine whether the presence or absence of RamA 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 3 we can see that in the presence of RamA there is increased GFP fluorescence suggesting that the acrRA promoter is being activated by RamA.
+https://static.igem.org/mediawiki/2016/d/dd/T--Dundee--Results11.png
+Figure 1:  pUniprom-ramA was transformed into Rosetta cells containing a viral T7 polymerase which is activated by IPTG. Anti-HA tag western blot for RamA in pUniprom within the Rosetta cells (DE3). Cells were subcultured from an overnight at 37OC. Once an OD of 0.4 was reached the samples were added to a final concentration of 1mM IPTG. 1ml of this was pelleted and Laemlli buffer added before loading 15 µl samples onto the protein gel. SDS PAGE (12% acrylamide) was run and transferred to PVDF membrane followed by probing with anti-HA antibody.
+https://static.igem.org/mediawiki/2016/1/1b/T--Dundee--GreenResults2.png
+Figure 2: Microscopy fluorescence imaging for PacrRA-gfp with and without RamA transcription factor on MacConkey agar plates.
+https://static.igem.org/mediawiki/parts/3/3e/RamA-frulhuq.png
+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.
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