Difference between revisions of "Part:BBa K1350006"
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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.  
 
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.  
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'''  ===Verification of Mdfa==='''
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===Verification experiment on Mdfa===
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<center>[[File:电泳.png|200px]]
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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.
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<br><center>[[File:Kil_1.jpg|160px]]
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Figure 1.Determination of enzyme activity in cell supernatant and cells<br>
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Lane 1.enzyme activity of cell supernatant with kil<br>
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Lane 2.enzyme activity of cells with kil<br>
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Lane 3.enzyme activity of cell supernatant without kil<br>
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Lane 4.enzyme activity of cells without kil<br>
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[[File:Kil_2.jpg|100px]]
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<br>Figure 5.Determination of enzyme activity in cell supernatant<br>
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Lane 1.enzyme activity of cell supernatant without kil<br>
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Lane 3.enzyme activity of cell supernatant with kil</center><br>
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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.
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The above figures show that the E-coli transformed Mdfa proves to grow more normally than none Mdfa E-coli.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.
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1350006 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1350001 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1350006 parameters</partinfo>
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<partinfo>BBa_K1350001 parameters</partinfo>
 
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Revision as of 17:28, 14 October 2014

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.

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.


Kil 1.jpg

Figure 1.Determination of enzyme activity in cell supernatant and cells
Lane 1.enzyme activity of cell supernatant with kil
Lane 2.enzyme activity of cells with kil
Lane 3.enzyme activity of cell supernatant without kil
Lane 4.enzyme activity of cells without kil

Kil 2.jpg
Figure 5.Determination of enzyme activity in cell supernatant
Lane 1.enzyme activity of cell supernatant without kil

Lane 3.enzyme activity of cell supernatant with kil

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 above figures show that the E-coli transformed Mdfa proves to grow more normally than none Mdfa E-coli.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.


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]