Difference between revisions of "Part:BBa K3146011"
 
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This is a composite part which Includes a promoter (BBa_J23100), RBS (BBa_B0034) and CRP (BBa_K3146004)
 
This is a composite part which Includes a promoter (BBa_J23100), RBS (BBa_B0034) and CRP (BBa_K3146004)
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cAMP receptor protein involved in the iron homeostasis. The expression of many anaerobic respiratory genes in MR-1, including those for the reduction of fumarate, dimethyl sulfoxide, and metal oxides, is regulated by cyclic AMP receptor protein (CRP). CRP i coordinately regulates the expression of catabolic and respiratory pathways in MR-1, including d-lactate dehydrogenase and anaerobic terminal reductases. CRP directly binds to the upstream regions of omcA and mtrC in the presence of cAMP, and activates the transcription of these metal-reduction genes.
  
 
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<partinfo>BBa_K3146011 parameters</partinfo>
 
<partinfo>BBa_K3146011 parameters</partinfo>
 
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Shewanella oneidensis strain MR-1 has been been extensively studied due to it’s extracellular electron transfer system(MTR pathway) under anaerobic conditions. We have assayed wether over expression of the following genes affected the Microbial Fuel Cells(MFC ) output following genes were encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP), the ex-tracytoplasmic function sigma factor RpoE, oxygen-responsive transcriptional regulator Fnr (EtrA) and the key gene responsible for rhamnolipids synthesis (rhlA).
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All genes were Ligated to BBa_J23100 constitutive promoter, BBa_B0034 strong Ribosome Binding Site and transformed into Shewanella oneodensis in Psb3C1 vector as described in Shewanella Conjugation protocol. Ligations were performed using 3A assembly, Psb1C3 vector was used as Shewanella oneidensis Mr-1 is naturally resistant to Ampicillin. Cultures were grown in double antibiotic broth(100ng/ul of Chlorampheniucol and 100ng/ul Ampicillin) for 18 hours prior to inoculation of MFCs. 6 MFCs were set up as described in MFC protocol including  control A Shewanella MR-1 without antibiotic, control B Shewanella Oneidensis MR-1 containing TPHP-PSB1C3(terephthalic acid permease channel) and 4 MFC’s test constructs supplemented with 100ng/ul of Chlorampheniucol and 100ng/ul Amphicillin for selection: CRP, EtrA, rhlA, RpoE. MFC out data was collected over 4 days.
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[[File:T--Westminster UK--FinalMFC-graph.jpeg|404px|thumb|center|Figure 1]]
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Out of all constructs tested, over expression of oxygen-responsive transcriptional regulator Fnr EtrA showed most significant increase compared to both controls as can be seen in Figure 1. Interestingly, Control Shewanella Oneidensis containing TPHP-PSB1C3(terephthalic acid permease channel) showed significantly higher output compared to control as well as CRP, Rpoe and RhlA. Overexprerssion of RpoE and RhlA were not successful at increasing MFC output comparing to both controls and Overexpression of CRP in Shewanella produced higher output than one out of 2 controls.
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This work could be build upon to study MTR pathway and anaerobic response in S. Oneidensis MR-1 and further increase efficiency of Microbial Fuel Cells as renewable energy source. Over-expression of anaerobic response transcription factors in S. Oneidensis could solve the problem of anaerobic requirements and oxygen leakage in Microbial fuel cells, however further tests are required to confirm functionality of the constructs in various conditions.

Latest revision as of 03:35, 22 October 2019


CRP with promoter and RBS

This is a composite part which Includes a promoter (BBa_J23100), RBS (BBa_B0034) and CRP (BBa_K3146004)

cAMP receptor protein involved in the iron homeostasis. The expression of many anaerobic respiratory genes in MR-1, including those for the reduction of fumarate, dimethyl sulfoxide, and metal oxides, is regulated by cyclic AMP receptor protein (CRP). CRP i coordinately regulates the expression of catabolic and respiratory pathways in MR-1, including d-lactate dehydrogenase and anaerobic terminal reductases. CRP directly binds to the upstream regions of omcA and mtrC in the presence of cAMP, and activates the transcription of these metal-reduction genes.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 88
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Shewanella oneidensis strain MR-1 has been been extensively studied due to it’s extracellular electron transfer system(MTR pathway) under anaerobic conditions. We have assayed wether over expression of the following genes affected the Microbial Fuel Cells(MFC ) output following genes were encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP), the ex-tracytoplasmic function sigma factor RpoE, oxygen-responsive transcriptional regulator Fnr (EtrA) and the key gene responsible for rhamnolipids synthesis (rhlA).

All genes were Ligated to BBa_J23100 constitutive promoter, BBa_B0034 strong Ribosome Binding Site and transformed into Shewanella oneodensis in Psb3C1 vector as described in Shewanella Conjugation protocol. Ligations were performed using 3A assembly, Psb1C3 vector was used as Shewanella oneidensis Mr-1 is naturally resistant to Ampicillin. Cultures were grown in double antibiotic broth(100ng/ul of Chlorampheniucol and 100ng/ul Ampicillin) for 18 hours prior to inoculation of MFCs. 6 MFCs were set up as described in MFC protocol including control A Shewanella MR-1 without antibiotic, control B Shewanella Oneidensis MR-1 containing TPHP-PSB1C3(terephthalic acid permease channel) and 4 MFC’s test constructs supplemented with 100ng/ul of Chlorampheniucol and 100ng/ul Amphicillin for selection: CRP, EtrA, rhlA, RpoE. MFC out data was collected over 4 days.

Figure 1

Out of all constructs tested, over expression of oxygen-responsive transcriptional regulator Fnr EtrA showed most significant increase compared to both controls as can be seen in Figure 1. Interestingly, Control Shewanella Oneidensis containing TPHP-PSB1C3(terephthalic acid permease channel) showed significantly higher output compared to control as well as CRP, Rpoe and RhlA. Overexprerssion of RpoE and RhlA were not successful at increasing MFC output comparing to both controls and Overexpression of CRP in Shewanella produced higher output than one out of 2 controls.

This work could be build upon to study MTR pathway and anaerobic response in S. Oneidensis MR-1 and further increase efficiency of Microbial Fuel Cells as renewable energy source. Over-expression of anaerobic response transcription factors in S. Oneidensis could solve the problem of anaerobic requirements and oxygen leakage in Microbial fuel cells, however further tests are required to confirm functionality of the constructs in various conditions.