Part:BBa_K1350006

promoter+RBS(BBa_K608002)+Mdfa

The resistance of Escherichia coli to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs);MdfA,the known bacterial MDR transporters probably utilize the membrane potential(Δψ) and/or the proton gradient (ΔpH) as the driving force for drug export.In Escherichia coli, Mdfa are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress.^(1,2,3)

This process is mainly activated by the cross membrane proton electrochemical gradient .changing the electrochemical electric potential of proton built by respiration chain to the cross membrane Na+ electrochemical potential. From research about the Na+/ H+ antiporter of E.coli, mdfa can pump Na+ out of cell when it pumps H+ into cell and ATP is consumed through the process.

Verification experiment on Mdfa

After primer design, pcr and gene sequencing, we linked Mdfa to a strong promoter and RBS(BBa_K608002). Then the product, Mdfa device(BBa_K1350006) has been done.The results shows in the figure below.^(5,6)

Figure 1. Restriction digestion plasmid gel picture

Figure 2.Growth contrast in high pH picture

Culture four concentration gradients of E-coli (Mdfa transformed) and E-coli (BBa_K608002 transformed) in high pH conditions. The dilution proportion from left to right respectively is zero, doubling,10-fold,100-fold. The E-coli transformed Mdfa proves to grow more normally than none Mdfa E-coli.

Figure 3. Chart of OD value of Mdfa device and control group in different pH

Using OD values to validate the growth of E-coli (with Mdfa) and E-coli (with a promoter and RBS). Compared with the E-coli with a promoter, E-coli with Mdfa grow more actively in pH9.0 to pH9.5. In the pH9.75 to pH10, none of them grow.

Result

The result shows Mdfa does work actively in E-coli ,helping tolerate the high pH conditions in a range from pH 9.0 to Ph 9.5.

Reference

1. Bolhuis, H., G. Poelarends, H. W. van Veen, B. Poolman, A. J. M. Driessen,and W. N. Konings. 1995. The lactococcallmrP gene encodes a protonmotive force-dependent drug transporter. J. Biol. Chem. 270:26092–26098.2. Grinius, L. L., and E. B. Goldberg. 1994. Bacterial multidrug resistance is due to a single membrane protein which functions as a drug pump. J. Biol.Chem. 269:29998–30004.3. Grinius, L. L., and E. B. Goldberg. 1994. Bacterial multidrug resistance is due to a single membrane protein which functions as a drug pump. J. Biol.Chem. 269:29998–30004.4. Etana Padan and Eitan Bibi.. Alkaline pH homeostasis in bacteria: New insights. 2005Nov;1717(2):67-885.MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition.Edgar R1, Bibi E. J Bacteriol. 1997 Apr;179(7):2274-80.6. mdfA multidrug efflux system protein [ Escherichia coli str. K-12 substr. MG1655 ]. NC_000913.3

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 552
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]