Device

Part:BBa_K1350006

Designed by: Yiting Luo   Group: iGEM14_SZU-China   (2014-09-28)
Revision as of 15:26, 15 October 2014 by Yitingluo (Talk | contribs)

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.


电泳.png

Figure 1. Restriction digestion plasmid gel picture

PH7.jpgPH9.png
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.


Ph9 副本.pngPh9.25 副本.pngPh9.5.png Ph9.75 副本.pngPh10 副本.png


Figure 3. Chart of OD value of Mdfa device and control group in different pH
</center> 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.</center>

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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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