Team:BGIC-Global/Experiment Design
How can we detect formaldehyde?
We need:
A formaldehyde induced promoter -- pFrmR
E. coli has a native formaldehyde-inducible promoter (pFrm), 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.16 7 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 a far lesser degree, and not at all to a range of other aldehydes and alcohols tested.16, 17 10 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.
Based on the research of Rohlhill J. et al., we had an engineered formaldehyde-inducible promoter synthesized, which is called pFrmR, and has been proved to retain formaldehyde responsiveness, with 2-fold higher GFP expression in response to 100 μM formaldehyde than the native pFrm.
A reporter system – EGFP
EGFP is a basic (constitutively fluorescent) green fluorescent protein published in 1996, derived from Aequorea victoria. It is reported to be a rapidly-maturing weak dimer with moderate acid sensitivity.
A formaldehyde induced promoter -- pFrmR
E. coli has a native formaldehyde-inducible promoter (pFrm), 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.16 7 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 a far lesser degree, and not at all to a range of other aldehydes and alcohols tested.16, 17 10 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.
Based on the research of Rohlhill J. et al., we had an engineered formaldehyde-inducible promoter synthesized, which is called pFrmR, and has been proved to retain formaldehyde responsiveness, with 2-fold higher GFP expression in response to 100 μM formaldehyde than the native pFrm.
A reporter system – EGFP
EGFP is a basic (constitutively fluorescent) green fluorescent protein published in 1996, derived from Aequorea victoria. It is reported to be a rapidly-maturing weak dimer with moderate acid sensitivity.
How can we remove formaldehyde?
We need:
A functional part which could be induced by formaldehyde -- GFA
Gfa is a zinc-dependent enzyme that accelerates the spontaneous the condensation of GSH with formaldehyde to form HMGS. It plays an important role in the the best characterized pathway for formaldehyde detoxification.
A functional part which could be induced by formaldehyde -- GFA
Gfa is a zinc-dependent enzyme that accelerates the spontaneous the condensation of GSH with formaldehyde to form HMGS. It plays an important role in the the best characterized pathway for formaldehyde detoxification.
Initiate Experiment Design
The exogenous gene gfa was introduced into E. coli BL21 to further enhance the endogenous pathway of E. coli BL21 to degrade formaldehyde, thereby realizing the purpose of using the engineered bacteria to degrade formaldehyde in the environment.
At the same time, EGFP will not only act as a reporter to formaldehyde, it will also indirectly reflect the degradation efficiency of formaldehyde through green fluorescence intensity. That is, the expression of the EGFP is initiated by a modified formaldehyde-inducible promoter, pFrmR. Based on the above purposes, we will construct the two vectors, as shown above. The two plasmids will be separately introduced into E. coli BL21, and then the two different vectors will be mixed in a certain ratio to carry out an experiment on formaldehyde detection and degradation. Furtherly, these 2 separate parts could be combined into one circuit and constructed on the same plasmid:
At the same time, EGFP will not only act as a reporter to formaldehyde, it will also indirectly reflect the degradation efficiency of formaldehyde through green fluorescence intensity. That is, the expression of the EGFP is initiated by a modified formaldehyde-inducible promoter, pFrmR. Based on the above purposes, we will construct the two vectors, as shown above. The two plasmids will be separately introduced into E. coli BL21, and then the two different vectors will be mixed in a certain ratio to carry out an experiment on formaldehyde detection and degradation. Furtherly, these 2 separate parts could be combined into one circuit and constructed on the same plasmid:
Improved Experiment Design – Round 1
When we were waiting for our synthesized plasmids, we did more research which led to the update of our system design. We improved the responding system, which is shown above. The renewed formaldehyde responding system could simulate intrinsic negative feedback system, FrmRAB, in E. coli B21. In other words, 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.
Improved Experiment Design – Round 2
In addition, by replacing EGFP with Nanoluc, luminescence could be observed with the naked eye. We had this idea after thoroughly analyze the (potential) customer survey and talk to different stakeholders. And the below figure shows how the bacteria that we provide in our product will work as a sensor and remover of formaldehyde:
Improved Experiment Design – Round 3 (Future Plan)
To optimize our experiment result, we referred to the research of Rohlhill J et. al on the activity of pFrmR, and plan to use lactose-inducing promotor to operate expression of FrmR inhibiting factor separately.
Experiment Devise
