Difference between revisions of "Part:BBa K2728004"
(→‎Experimental Characterization)
Line 29: Line 29:
 
<br />
 
<br />
 
=== Experimental Characterization ===
 
=== Experimental Characterization ===
−
We have tested the influence of GFA in different formaldehyde concentration. Cultures were inoculated from overnights to an optical density (OD) of 0.05 in 5 mL in 15 mL disposable culture tubes, incubated at 37 °C for 2−3 h, and dosed with 0−500 μM formaldehyde (MP Biomedicals, Santa Ana, CA). And we aimed at comparing the effect of GFA test E. coli with normal E. coli BL21 in formaldehyde environment by OD600nm. But in 100~500uM formaldehyde, there are no difference in BL21 with test E. coli.
+
Culture environment:  saturated formaldehyde aqueous solution with concentration of 37 percent<br />
 +
Instruments: tecan infinite m1000<br />
 +
Experiment group: BL21 with pFrmR+GFP and BL21 with GFA in ratio of 1:1<br />
 +
Control group A: BL21<br />
 +
Control group B: BL21 with pFrmR+EGFP<br />
 
<br />
 
<br />
 +
#Test the OD number of overnight-cultured strain;
 +
#Diluted to 0.05, the strains are cultured under 37 celsius, 220prm.
 +
For control groups:<br />
 +
#Take 5 tubes of 1ml system both from the control groups and add 0.5ul saturated formaldehyde solution into each tube. Mix evenly.
 +
For experiment group:<br />
 +
#Take 500ul from each strain and mix them together.
 +
#Add 0.5ul of saturated formaldehyde aqueous solution.
 
<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--GFA.png|left|border|500px]]<br clear=all>
 +
The figure indicates that the experiment expressed the highest amount of fluorescence.<br />
 +
The experiment only had half amount of EGFP bacteria compared with the control group B, but produced higher expression. The only way to explain this result is that GFA played an important role to degrade the formaldehyde, therefore decreased the toxicity of the environment of bacteria and enhance the survival rate. This also indicates that our engineered system shown below works. Due to the time limitation, we only successfully finished only one trial. We will test more combination of mixed ratio and formaldehyde concentrations in the coming days.
 +
<br />
 +
[[File:T--BGIC-Global--circuitmix.png|left|border|800px]]<br clear=all>
 +
<br />
 +
<br />
 +
 
=== Parts Verification Before Submission ===
 
=== 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.=)
 
We verified our parts in the lab before submission. They are reliable! Please feel free to apply them onto your project.=)

Revision as of 17:48, 17 October 2018


GFA - A Glutathione-Dependent Formaldehyde-Activating Enzyme

Basic Description

An enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction, which catalyze the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene GFA is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. The glutathione-dependent formaldehyde conversion to formate starts with the adduct formation, formaldehyde reacts with the SH group of glutathione producing S-hydroxymethylglutathione (Reaction 1).

caption

Fig 1: Thiol-dependent pathway


Formaldehyde-converting enzymes-Gfa is composed of one type of subunit of about 20 kDa and lack a chromophoric prosthetic group. In addition, both enzymes are encoded next to genes for enzymes involved in further oxidation of the cofactor-bound one-carbon unit to carbon dioxide.

caption

Fig 2: 3D structure (from www.uniprot.org)



Sequence

We ordered the synthesized plasmid from Genscript. After restriction digestion & transformation, we got our E. coli with GFA.

T--BGIC-Global--gfasyn1.png

caption

Fig 3: Order info



Origin

This gene originated from P. denitrificans. Putative proteins with sequence identity to GFA from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.

Experimental Characterization

Culture environment: saturated formaldehyde aqueous solution with concentration of 37 percent
Instruments: tecan infinite m1000
Experiment group: BL21 with pFrmR+GFP and BL21 with GFA in ratio of 1:1
Control group A: BL21
Control group B: BL21 with pFrmR+EGFP

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

For control groups:

  1. Take 5 tubes of 1ml system both from the control groups and add 0.5ul saturated formaldehyde solution into each tube. Mix evenly.

For experiment group:

  1. Take 500ul from each strain and mix them together.
  2. Add 0.5ul of saturated formaldehyde aqueous solution.


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--GFA.png

The figure indicates that the experiment expressed the highest amount of fluorescence.
The experiment only had half amount of EGFP bacteria compared with the control group B, but produced higher expression. The only way to explain this result is that GFA played an important role to degrade the formaldehyde, therefore decreased the toxicity of the environment of bacteria and enhance the survival rate. This also indicates that our engineered system shown below works. Due to the time limitation, we only successfully finished only one trial. We will test more combination of mixed ratio and formaldehyde concentrations in the coming days.

T--BGIC-Global--circuitmix.png



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. Gonzalez, C. F., Proudfoot, M., Brown, G., Korniyenko, Y., Mori, H., Savchenko, A. V., & Yakunin, A. F. (2006). Molecular Basis of Formaldehyde Detoxification. Journal of Biological Chemistry, 281(20), 14514–14522.
  2. Goenrich, M., Bartoschek, S., Hagemeier, C. H., Griesinger, C. & Vorholt, J. A. (2002). A glutathione-dependent formaldehydeactivating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two-dimensional proton exchange NMR spectroscopy.J Biol Chem 277, 3069–3072.
  3. 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]