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Results

P-coumaric acid

We managed to clone out and biobrick tyrosine ammonia lyase and verify the biobrick by sequencing. Also we did succeful characterization on this part, showing that it works as expected. We managed to express our enzyme and detect it in a western blot, and also detect our metabolite in both spectrophotometry and hplc. The biobrick was characterized in E. coli d5 alpha and E. coli nissle. For detailed information about the characterization methods, see the protocol section. To read more about P-coumaric acid, click here

1. Positive control
2. TAL with Cp8 promoter
3. TAL with J23110 promoter
4. Negative control

Figure 1:SDS-page and western blot. Expression of Tyrosine ammonia lyase with constitutive promoters. The negative control is empty, showing that there is no natural protein in E. coli with the same attributes.

Figure 2:Absorbance spectra of extracts collected from bacterial cultures. Samples were collected 21 h and 48 h after 30 °C incubation. The negative control is an extract from a strain with no TAL gene on transformed plasmid. The positive control is an extract a culture of the same strain as the negative control but with added p-coumaric acid to a concentration of 500 µM before extraction. P-coumaric acid absorbance spectra has two peaks. The one around 305 nm is preferable to detect because of background from bacteria. (a) Spectra from the strain with TAL CDS with promoter J23110.(b) Spectra from the strain with TAL CDS with promoter CP8. (c) Spectra from the strain with TAL CDS with promoter J23101.

	Figure 3: Graph showing the HPLC result of a sample prepared from E. coli expressing tyrosine ammonia lyase. Reverse phase HPLC with a C18 matrix was used. The peak for p-coumaric acid can be seen ~10 min, as shown by the standard sample below.
	Figure 4: Graph showing the HPLC result of a sample standard with p-coumaric acid.
	Figure 5: E. coli culture injected to the hplc without our biobrick tyrosine ammonia lyase. Here we can see that there is originally no peak at 10 minutes.