Part:BBa_K1423006
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. Because isoamyl acetate is produced in response to the presence of a heavy metal, bacteria transformed with the construct can be used as a reporter for heavy metal contamination.
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 enzyme. The conversion of isoamyl alcohol to isoamyl acetate was calculated by measuring the area under the curve in the gas chromatograph. The results of this experiment can be seen by clicking on the link below.
![[1]](https://static.igem.org/mediawiki/2014/b/b6/GCMSresults%282%29.png){kind=link}
Cultures induced with 0.5 μM, 5 μM, and 10 μM sodium arsenite showed increased yield of isoamyl acetate over cultures that constitutively converts isoamyl alcohol to isoamyl acetate. Arsenite concentrations of 0.5 μM and 5 μM resulted in a yield that was over 3 times greater than that for the constitutive conversion to isoamyl acetate. The cultures induced with 10 μM arsenite produced about 2.5 times more isoamyl acetate than the culture constituitively expressing ATF1. Beyond sodium arsenite concentrations of 10 μM, isoamyl acetate production decreased because the cells could not withstand the high concentrations of arsenite.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 255
Illegal BglII site found at 646
Illegal BamHI site found at 1973 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1073
Illegal SapI.rc site found at 2081
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