Difference between revisions of "Part:BBa K1350006"

(Verification experiment on Mdfa)
Line 9: Line 9:
  
 
<br><center>[[File:Kil_1.jpg|160px]]
 
<br><center>[[File:Kil_1.jpg|160px]]
Figure 1.Determination of enzyme activity in cell supernatant and cells<br>
+
Figure 1. Restriction digestion plasmid gel picture<br>
Lane 1.enzyme activity of cell supernatant with kil<br>
+
Lane 2.enzyme activity of cells with kil<br>
+
Lane 3.enzyme activity of cell supernatant without kil<br>
+
Lane 4.enzyme activity of cells without kil<br>
+
  
 
[[File:Kil_2.jpg|100px]]
 
[[File:Kil_2.jpg|100px]]
<br>Figure 5.Determination of enzyme activity in cell supernatant<br>
+
<br>Figure 2.Growth contrast in high pH picture<br>
Lane 1.enzyme activity of cell supernatant without kil<br>
+
Line 1.E-coli with <br>
Lane 3.enzyme activity of cell supernatant with kil</center><br>
+
Line 2.enzyme activity of cell supernatant with kil</center><br>
  
 
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.  
 
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.  

Revision as of 17:35, 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. Restriction digestion plasmid gel picture

Kil 2.jpg
Figure 2.Growth contrast in high pH picture
Line 1.E-coli with

Line 2.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]