Difference between revisions of "Part:BBa K1962009"
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===Usage and Biology===
 
===Usage and Biology===
  
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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).
+
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 2 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.
+
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.
  
−
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.
+
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)..
  
  
−
https://static.igem.org/mediawiki/2016/1/1b/T--Dundee--GreenResults2.png
+
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: Microscopy fluorescence imaging for PacrRA-gfp with and without RamA transcription factor on MacConkey agar plates.
 
  
 +
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/d/dd/T--Dundee--Results11.png
 
  
−
Figure 2: 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.
  
  

Revision as of 23:53, 22 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.


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.


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.


T--Dundee--GreenResults2.png

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


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.


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]