Part:BBa_K3146013

RpoE with promoter and RBS

This is a composite part which Includes a promoter (BBa_J23100), RBS (BBa_B0034) and RpoE (BBa_K3146009)

RNA polymerase, extracytoplasmic transcription factor E found in Shewanella oneidensis MR-1 strain. Involved as an aerobic response transcription factor.

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.

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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]