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BACKGROUND
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 (1, 2, 3).
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. (4)
Secondary structure model of MdfA (5). TMSs of 19 to 21 hydrophobic amino acid residues were chosen based on the hydropathyprofile. Positively charged residues are in bold-face type, and the transport protein’s consensus footprint (11, 14) is emphasized in the loop between TMS 2 and TMS3 (numbers at top of panel C indicate TMSs).
METHOD
The sequence of Mdfa was from GenBank. 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. (6) We both single digested and double digested the Mdfa+pSB1C3(BBa_K1350005) and promoter+RBS+Mdfa+pSB1C3(BBa_K1350006)to testify if we have inserted the Mdfa and promoter+RBS +Mdfa successfully.
RESULT
Figure 1. Restriction digestion plasmid gel picture
Mdfa is known as 1233 bp.The Mdfa part from double digestion of Mdfa+pSB1C3 is smaller than the promoter+RBS+Mdfa part from double digestion of promoter+Mdfa+pSB1C3.
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
Conclusion
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.
DISCUSSION
BBa_K608002 by iGEM11_Freiburg 2011 is a strong Promoter and strong RBS and it is not related to tolerating alkaline stress, so it will be a good no-treatment control group.REFERENCES
- 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 proton motive 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.
- . Etana Padan and Eitan Bibi.. Alkaline pH homeostasis in bacteria: New insights. 2005 Nov;1717(2):67-88
- 5.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
