Part:BBa_K1465107

porine fwd

---

Primer porine fwd (BBa_K1465107) is part of the construction of an electrophilic E. coli strain, which metabolism could be increased by growing under influence of electric power. It is part of a primer collection containing BBa_K1465107, BBa_K1465108, BBa_K1465109 and BBa_K1465110. The aim is to create a deletion cassette for knocking out C4 carboxylate transporter DcuB and insert outer membrane porine OprF (BBa_K1172507) into E. coli chromosome in the same step.

Usage and Biology

---

C4 Carboxylate Antiporter DcuB

Under anaerobic conditions E. coli cells use different alternative electron acceptors instead of oxygen. Partially the bacteria use fumarate respiration, whereby fumarate is reduced into succinate. There are also other potential less-oxidizing substances for bacteria to release their electrons, for example anorganic compounds like nitrate (NO₃^-) or sulfate (SO₄^2-).(Gottschalk et al., 1986) Fumarate respiration leads to succinate excretion through the C4 carboxylate transporter DcuB. It is an antiporter which exchanges fumarate against succinate under anaerobic conditions. Under aerobic condition there is usually no succinate release observed. In connection to the carbon dioxide fixation in our second module we planned on working under oxygen limiting conditions, hence effective carbon dioxid fixation is possible. So in case of oxygen limiting conditions, there could occured partial fumarate respiration in E. coli. Besides there was shown activity of DcuB antiporter in the presence of high fumarate concentrations (Janausch, 2001). To achieve an effective electron uptake and prevent any succinate excretion, the C4 carboxylate antiporter DcuB has to be knocked out in our E. coli strain.
We planned a targeted knockout of the dcuB gene in E. coli KRX using the Genebridge Red/ET-System. In the same step we are going to integrate the outer membrane porine OprF (BBa_K1172507) into the bacterial chromosome under controll of a constitutive promotor (BBa_J23104). This ensure the permeability of the outer membrane and avoid a plasmid overload of the bacteria, because for our system the outer membrane porines are indispensable.

Sequence and Features

---

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 57
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Results

For detailed information see BBa_K1465110