Difference between revisions of "Part:BBa K1919233"

Revision as of 16:13, 19 October 2016

AarC coding sequence

Succinyl-CoA:Acetate CoA-Transferase(AarC) can let acetate and Succinyl-CoA become acetyl-CoA and succinate. E.coli can use acetate as energy sources by this enzyme. This enzyme also can give E.coli some ability of anti acetic acid. We made five devices for constitutive expressing AarC enzyme. These devices have different promoter. From strong to weak are BBa_K1919200 (J23100), BBa_K1919204 (J23104), BBa_K1919206 (J23106), BBa_K1919205 (J23105) and BBa_K1919214 (J23114). All of these expression devices have His-tag at C-terminal.

Usage and Biology

After we get the expression device, we try to detect the activity of AarC. Subject to conditions, we can't use the electrospray ionization-mass spectrometry as same as original document, so we used high performance liquid chromatography (HPLC) to measuring the concentration of acetic-acid and acetate. Unfortunately, LB has a very complex compound and there is a peak at the same location of acetate/acetic-acid. Therfore, we have to added 2mg/mL acetate/acetic-acid then we can detect them clearly. But this led to some bad situations. Because the background noise of LB medium, quantitative analysis is not valid. On the other hand, high concentration of acetic-acid have a negative effect of E.coli.

Utilization of acetate

Figure 1: The result of HPLC, control group.The peak at 2.908 represent acetate. Peak area is 3149002

Figure 2: The result of HPLC, promotor J23100, the strongest one.The peak at 2.966 represent acetate. Peak area is 1443872

Figure 3: The result of HPLC, promotor J23114, the weakest one.The peak at 2.915 represent acetate. Peak area is 2142511

The peak area means relative concentration of acetate. Obviously, AarC have a good ability of using acetate and it worked.

Resistance of acetic acid

Figure 4: The result of HPLC, control group.The peak at 2.871 represent acetic acid. Peak area is 2816616

Figure 5: The result of HPLC, promotor J23100, the strongest one.The peak at 2.794 represent acetic acid. Peak area is 5664716

Figure 6: The result of HPLC, promotor J23114, the weakest one.The peak at 2.851 represent acetic acid. Peak area is 5382006

The peak area means relative concentration of acetic acid. We can see the concentration of acetic acid doubled. From a macro perspective, E.coli didn't growth at LB medium have 2mg/mL acetic acid. We guess e.coli dead and released AarC. AarC played a reverse role: It let acetyl coenzyme A become acetic acid, so there are more acetic acid than control group. We also need more experiments to verify the conjecture.

Sequence and Features