Difference between revisions of "Part:BBa K2728001"

(→‎Experimental Characterization)
(→‎Experiment 1)
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<br /><br />
 
<br /><br />
 
===== Experiment 1 =====
 
===== Experiment 1 =====
−
Culture environment: saturated formaldehyde aqueous solution with concentration of 37 percent.<br />
+
Culture environment: saturated formaldehyde aqueous solution with concentration of 37 percent<br />
 
Instruments: tecan infinite m1000<br />
 
Instruments: tecan infinite m1000<br />
−
Experiment group: BL21 with pFrmR+GFP<br />
+
Experiment group: BL21 with pFrmR+EGFP<br />
 
Control group: BL21 (with no plasmid transformed)<br />
 
Control group: BL21 (with no plasmid transformed)<br />
 
<br />
 
<br />
Line 48: Line 48:
 
Results:<br />
 
Results:<br />
 
[[File:T--BGIC-Global--pfmxrxp1.png|left|border|800px]]<br clear=all>
 
[[File:T--BGIC-Global--pfmxrxp1.png|left|border|800px]]<br clear=all>
−
 
 
<br />
 
<br />
 +
[[File:T--BGIC-Global--pfmxrxp2.png|left|border|500px]]<br clear=all>
 +
<br />
 +
In solution with 1ul of formaldehyde, luminescence was the most obvious, indicating more active pFrmR.
 +
<br />
 +
<br />
 +
===== Experiment 2 =====
 +
Culture environment:  saturated formaldehyde aqueous solution with concentration of 37 percent<br />
 +
Instruments: tecan infinite m1000<br />
 +
Experiment group: BL21 with pFrmR+EGFP<br />
 +
Control group: BL21<br />
 +
<br />
 +
#Test the OD number of overnight-cultured strain;
 +
#Diluted to 0.05, the strains are cultured under 37 celsius, 220prm.
 +
For experiment group:<br />
 +
#Take 5 tubes of 1ml system both from experiment group and control group and add 0ul, 1ul, 2.5ul, 5ul, and 7.5ul saturated formaldehyde solution into each tube. Mix evenly.
 +
#Take 150ul from each of the tubes into 96 orifice plate.
 +
#Test fluorescence in tecan infinite m1000.
 +
<br />
 +
<br />
 +
Related conditions:<br />
 +
Excitation Wavelength 495 nm<br />
 +
Emission Wavelength 525 nm<br />
 +
List of actions in this measurement script:<br /> 　
 +
Shaking (Orbital) Duration: 900 s<br />
 +
Shaking (Orbital) Amplitude: 2 mm<br />
 +
Shaking (Orbital) Frequency: 306 rpm<br />
 +
[[File:Pfmxrxp3.png|left|border|800px]]<br clear=all>
 +
<br />
 +
Results:<br />
 +
[[File:T--BGIC-Global--pfmxrxp4.png|left|border|500px]]<br clear=all>
 +
Summarized graph for all groups of FrmR and BL21. In solution with 1ul of formaldehyde, luminescence was the most obvious, indicating more active pFrmR.<br />
 +
<br />
 +
[[File:T--BGIC-Global--pfmxrxp5.png|left|border|500px]]<br clear=all>
 +
In solution with no formaldehyde dosed, BL21 with EGFP had similar expression of fluorescence with control group.<br />
 +
<br />
 +
[[File:T--BGIC-Global--pfmxrxp6.png|left|border|500px]]<br clear=all>
 +
In solution with addition of 0.2ul of formaldehyde, BL21 with EGFP had similar expression of fluorescence with the control group, which means in this condition the activity of pFrmR was low.<br />
 +
<br />
 +
[[File:T--BGIC-Global--pfmxrxp7.png|left|border|500px]]<br clear=all>
 +
In solution with addition of 0.5ul of formaldehyde, the difference in fluorescent degree between BL21 with EGFP and the control group was clear, indicating higher activity of pFrmR.<br />
 +
<br />
 +
[[File:T--BGIC-Global--pfmxrxp8.png|left|border|500px]]<br clear=all>
 +
In solution with addition of 1ul of formaldehyde, the difference in fluorescent degree between BL21 with EGFP and the control group was the most obvious, indicating highest activity of pFrmR which successfully activated EGFP.<br />
 +
<br />
 +
[[File:T--BGIC-Global--pfmxrxp9.png|left|border|500px]]<br clear=all>
 +
In solution with addition of 1.5ul formaldehyde solution, the difference in fluorescent degree between BL21 with EGFP and the control group was clear, indicating the certain level of activity of pFrmR.<br />
 +
<br />
 +
[[File:T--BGIC-Global--pfmxrxp10.png|left|border|500px]]<br clear=all>
 +
In solution with addition of 1ul of formaldehyde solution, the activity of pFrmR was the highest.<br />
 +
<br />
 +
[[File:T--BGIC-Global--v2exd.png|left|border|500px]]<br clear=all>
 +
In formaldehyde free condition, FrmR inhibiting factor regulates pFrmR through negative feedback, lowering the activity of the promotor. After adding the inductor, FrmR inhibiting factor combined with formaldehyde molecules, lowering the effect of the negative feedback, thus the activity of pFrmR will be higher.<br />
 
<br />
 
<br />
  

Revision as of 17:17, 17 October 2018


pfrmR - An Engineered Formaldehyde-Inducible Promoter

Basic Description

This promoter is an engineered formaldehyde-inducible promoter. Escherichia coli has a native formaldehyde-inducible promoter, pfrm, which is found upstream of the frmRAB formaldehyde detoxification operon. FrmR, the first product of the operon, is a member of the DUF156 family of DNA-binding transcriptional regulators. It binds the frmRAB promoter region and is negatively allosterically modulated by formaldehyde. FrmR is specific to formaldehyde, responding to acetaldehyde, methylglyoxal, and glyoxal to far lesser degrees and not at all to a range of other aldehydes and alcohols tested. The genes frmA and frmB encode a formaldehyde dehydrogenase and S-formylglutathione hydrolase, respectively, and are responsible for detoxifying formaldehyde to formic acid in a glutathione-dependent pathway. The negative-feedback regulation of the frmRAB operon is similar to that of many other prokaryotic operons, whereby the transcription factor represses its own transcription.

caption

Fig 1: Without Formaldehyde


caption

Fig 2: With Formaldehyde



Features

  1. It’s a formaldehyde-inducible promoter from E.coli.
  2. It’s an engineered promoter. It retains formaldehyde responsiveness, with 2-fold higher GFP expression in response to 100 μM formaldehyde than the native pfrm. Application of this promoter with higher basal and induced expression levels before methanol assimilation genes achieves higher biomass titers than the native E. coli pfrm.



Origins

Escherichia coli

Experimental Characterization

Though we failed a lot of times that we cannot even count, due to a lot of different reasons and controls. Eventually we found the proper setting of our equipment, and got some promising data as below:

Experiment 1

Culture environment: saturated formaldehyde aqueous solution with concentration of 37 percent
Instruments: tecan infinite m1000
Experiment group: BL21 with pFrmR+EGFP
Control group: BL21 (with no plasmid transformed)

  1. Test the OD number of overnight-cultured strain;
  2. Diluted to 0.05, the strains are cultured under 37 celsius, 220prm.
  3. Take 5 tubes of 1ml system both from experiment group and control group and add 0ul, 1ul, 2.5ul, 5ul, and 7.5ul saturated formaldehyde solution into each tube. Mix evenly.
  4. ake 150ul from each of the tubes into 96 orifice plate.
  5. est fluorescence in tecan infinite m1000.


Related conditions：
Excitation Wavelength 495 nm
Emission Wavelength 525 nm
List of actions in this measurement script:
　 Shaking (Orbital) Duration: 900 s
Shaking (Orbital) Amplitude: 2 mm
Shaking (Orbital) Frequency: 306 rpm

Results:

T--BGIC-Global--pfmxrxp1.png


T--BGIC-Global--pfmxrxp2.png


In solution with 1ul of formaldehyde, luminescence was the most obvious, indicating more active pFrmR.

Experiment 2

Culture environment: saturated formaldehyde aqueous solution with concentration of 37 percent
Instruments: tecan infinite m1000
Experiment group: BL21 with pFrmR+EGFP
Control group: BL21

  1. Test the OD number of overnight-cultured strain;
  2. Diluted to 0.05, the strains are cultured under 37 celsius, 220prm.

For experiment group:

  1. Take 5 tubes of 1ml system both from experiment group and control group and add 0ul, 1ul, 2.5ul, 5ul, and 7.5ul saturated formaldehyde solution into each tube. Mix evenly.
  2. Take 150ul from each of the tubes into 96 orifice plate.
  3. Test fluorescence in tecan infinite m1000.



Related conditions:
Excitation Wavelength 495 nm
Emission Wavelength 525 nm
List of actions in this measurement script:
　 Shaking (Orbital) Duration: 900 s
Shaking (Orbital) Amplitude: 2 mm
Shaking (Orbital) Frequency: 306 rpm

Pfmxrxp3.png


Results:

T--BGIC-Global--pfmxrxp4.png

Summarized graph for all groups of FrmR and BL21. In solution with 1ul of formaldehyde, luminescence was the most obvious, indicating more active pFrmR.

T--BGIC-Global--pfmxrxp5.png

In solution with no formaldehyde dosed, BL21 with EGFP had similar expression of fluorescence with control group.

T--BGIC-Global--pfmxrxp6.png

In solution with addition of 0.2ul of formaldehyde, BL21 with EGFP had similar expression of fluorescence with the control group, which means in this condition the activity of pFrmR was low.

T--BGIC-Global--pfmxrxp7.png

In solution with addition of 0.5ul of formaldehyde, the difference in fluorescent degree between BL21 with EGFP and the control group was clear, indicating higher activity of pFrmR.

T--BGIC-Global--pfmxrxp8.png

In solution with addition of 1ul of formaldehyde, the difference in fluorescent degree between BL21 with EGFP and the control group was the most obvious, indicating highest activity of pFrmR which successfully activated EGFP.

500px

In solution with addition of 1.5ul formaldehyde solution, the difference in fluorescent degree between BL21 with EGFP and the control group was clear, indicating the certain level of activity of pFrmR.

T--BGIC-Global--pfmxrxp10.png

In solution with addition of 1ul of formaldehyde solution, the activity of pFrmR was the highest.

T--BGIC-Global--v2exd.png

In formaldehyde free condition, FrmR inhibiting factor regulates pFrmR through negative feedback, lowering the activity of the promotor. After adding the inductor, FrmR inhibiting factor combined with formaldehyde molecules, lowering the effect of the negative feedback, thus the activity of pFrmR will be higher.

Improvements

The sequence of this part was taken from the research of Rohlhill J. et al. The 2 binding sites of variations are -35 and -10 (Fig 1). We ordered synthesized plasmid with pFrmR and EGFP from Gensceipt and constructed pFrmR-EGFP-FrmR reporter system on plasmid pUC57. After dosing formaldehyde of 100 to 400uM, we tested the EGFP expression to identify the activity of the formaldehyde induced response of this prompter.

The research conducted by Rohlhill J. et al. has proved that, pFrmR retains formaldehyde responsiveness, with 2-fold higher GFP expression in response to 100 μM formaldehyde than the native pfrm (BBa_K749008 )[4].This is quite a new discovery and we have not found other published researches having applied this promoter, which means, we are the first team that brings it to iGEM!

Future Improvements: We plan to optimize our reporter vector by introducing an independent promoter pLac to regulate the expression of FrmR.

T--BGIC-Global--pfrmr1.png

Fig 1: Sites of mutants


T--BGIC-Global--pfrmr2.png

Fig 2: Comparison of activity


T--BGIC-Global--pfrmr3.png

Fig 3: Current reporter system


T--BGIC-Global--pfrmr5.png

Fig 5: Future reporter system



Potential Application

  • To construct a formaldehyde sensor with this promoter.
  • To enable higher growth under formaldehyde pressure with the application of the engineered formaldehyde responsive promoter.



Parts Verification Before Submission

We verified our parts in the lab before submission. They are reliable! Please feel free to apply them onto your project.=)

T--BGIC-Global--partsub1.png

Fig 1: PCR (to get targeted genes)


T--BGIC-Global--partsub2.png

Fig 2: Restriction Digestion


T--BGIC-Global--partsub3.png

Fig 3: Ligation


T--BGIC-Global--partsub4.png

Fig 4: Colony PCR


T--BGIC-Global--partsub5.png

Fig 5: Gel Verification



References

  1. Osman, D., Piergentili, C., Chen, J., Sayer, L. N., Uson, I., Huggins, T. G., Robinson, N. J., and Pohl, E. (2016) The Effectors and Sensory Sites of Formaldehyde-Responsive Regulator FrmR and Metal-Sensing Variant. J. Biol. Chem. 291, 19502-19516
  2. Denby, K. J., Iwig, J., Bisson, C., Westwood, J., Rolfe, M. D., Sedelnikova, S. E., Higgins, K., Maroney, M. J., Baker, P. J., Chivers, P. T., and Green, J. (2016) The mechanism of a formaldehyde-sensing transcriptional regulator. Sci. Rep. 6, 38879
  3. Gonzalez, C. F., Proudfoot, M., Brown, G., Korniyenko, Y., Mori, H., Savchenko, A. V., and Yakunin, A. F. (2006) Molecular basis of formaldehyde detoxification: Characterization of two S-formylglutathione hydrolases from Escherichia coli, FrmB and YeiG. J. Biol. Chem. 281, 14514-14522
  4. Rohlhill J, Sandoval N R, Papoutsakis E T. Sort-seq approach to engineering a formaldehyde-inducible promoter for dynamically regulated Escherichia coli growth on methanol.[J]. Acs Synthetic Biology, 2017, 6(8)



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]