Difference between revisions of "Part:BBa K1423006"

Revision as of 15:48, 27 October 2014

Arsenic Inducible Banana Odor Generator

The arsenic inducible banana odor generator consists of the arsenic inducible promoter, J33201, and the composite part J45199, which is made up of a ribosome binding site, the ATF1 gene, and a double terminator. The ATF1 enzyme converts isoamyl alcohol to isoamyl acetate. E. coli transformed with this part can convert isoamyl alcohol to isoamyl acetate in the presence of heavy metals such as arsenic and cadmium.

To test the efficiency of the ATF1 enzyme when regulated by the arsenic inducible promoter, we grew a 50 mL culture of E. coli transformed with this Biobrick overnight in a 37°C shaker with 50 μL of chloramphenicol, then diluted the culture to OD 0.2 the following day. 2 mL of the diluted liquid culture were then added to 24 test tubes to create smaller liquid cultures. Varying concentrations of isoamyl alcohol and sodium arsenite were added to the 2mL cultures. In addition to the E. coli transformed with BBa_K1423006, a 10 mL liquid culture of E. coli transformed with BBa_K1423007 was grown overnight and diluted to OD 0.2. This diluted culture was then used to make 2 mL cultures in 6 test tubes. The contents of each test tube are outlined below. Note that three cultures were made for tubes 1-10.

1 a-c. 2mL E. coli transformed with BBa_K1423007

2 a-c. 2mL E. coli transformed with BBa_K1423007 + 5mM Isoamyl Alcohol

3 a-c. 2mL E. coli transformed with BBa_K1423006

4 a-c. 2mL E. coli transformed with BBa_K1423006 + 5mM Isoamyl Alcohol

5 a-c. 2mL E. coli transformed with BBa_K1423006 + 100 μM sodium arsenite

6 a-c. 2mL E. coli transformed with BBa_K1423006 + 5mM Isoamyl Alcohol + 0.5 μM sodium arsenite

7 a-c. 2mL E. coli transformed with BBa_K1423006 + 5mM Isoamyl Alcohol + 5 μM sodium arsenite

8 a-c. 2mL E. coli transformed with BBa_K1423006 + 5mM Isoamyl Alcohol + 10 μM sodium arsenite

9 a-c. 2mL E. coli transformed with BBa_K1423006 + 5mM Isoamyl Alcohol + 50 μM sodium arsenite

10 a-c. 2mL E. coli transformed with BBa_K1423006 + 5mM Isoamyl Alcohol + 100 μM sodium arsenite

11. 2mL LB + 5mM Isoamyl Alcohol + 5 mM Isoamyl acetate (standard)

12. 2mL 2x dilution of the standard

13. 2mL 10x dilution of the standard

14. 2mL 25x dilution of the standard

15. 2mL 50x dilution of the standard

The cultures above were parafilmed to prevent the evaporation of the isoamyl acetate product and were grown overnight in a 37°C shaker. Following the overnight incubation, 1 mL of each liquid culture was transfered into a gas chromatography vial. A gas chromatography/mass spectroscopy experiment was run to quantify the efficiency of the ATF1.

[1]

The link above leads to a quantification of the production of isoamyl acetate normalized to the same construct with the high strength constitutive promoter J23100.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 255
Illegal BglII site found at 646
Illegal BamHI site found at 1973
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1073
Illegal SapI.rc site found at 2081

@@ Line 7: / Line 7: @@
 The arsenic inducible banana odor generator consists of the arsenic inducible promoter, J33201, and the composite part J45199, which is made up of a ribosome binding site, the ATF1 gene, and a double terminator. The ATF1 enzyme converts isoamyl alcohol to isoamyl acetate. E. coli transformed with this part can convert isoamyl alcohol to isoamyl acetate in the presence of heavy metals such as arsenic and cadmium.
-To test the efficiency of the ATF1 enzyme when regulated by the arsenic inducible promoter, we grew a 50 mL  culture of E. coli transformed with this Biobrick overnight with 50 μL of chloramphenicol, then diluted the culture to OD 0.2 the following day. 2 mL of the diluted liquid culture were then added to 24 test tubes to create smaller liquid cultures. Varying concentrations of isoamyl alcohol and sodium arsenite were added to the 2mL cultures. In addition to the E. coli transformed with BBa_K1423006, a 10 mL liquid culture of E. coli transformed with BBa_K1423007 was grown overnight and diluted to OD 0.2. This diluted culture was then used to make 2 mL cultures in 6 test tubes. The contents of each test tube are outlined below. Note that three cultures were made for tubes 1-10.
+To test the efficiency of the ATF1 enzyme when regulated by the arsenic inducible promoter, we grew a 50 mL  culture of E. coli transformed with this Biobrick overnight in a 37°C shaker with 50 μL of chloramphenicol, then diluted the culture to OD 0.2 the following day. 2 mL of the diluted liquid culture were then added to 24 test tubes to create smaller liquid cultures. Varying concentrations of isoamyl alcohol and sodium arsenite were added to the 2mL cultures. In addition to the E. coli transformed with BBa_K1423006, a 10 mL liquid culture of E. coli transformed with BBa_K1423007 was grown overnight and diluted to OD 0.2. This diluted culture was then used to make 2 mL cultures in 6 test tubes. The contents of each test tube are outlined below. Note that three cultures were made for tubes 1-10.
 <p>1 a-c. 2mL E. coli transformed with BBa_K1423007</p>
@@ Line 25: / Line 25: @@
 <p>15. 2mL 50x dilution of the standard</p>
+The cultures above were parafilmed to prevent the evaporation of the isoamyl acetate product and were grown overnight in a 37°C shaker. Following the overnight incubation, 1 mL of each liquid culture was transfered into a gas chromatography vial. A gas chromatography/mass spectroscopy experiment was run to quantify the efficiency of the ATF1.