"
Team:Uppsala/results
From 2013.igem.org
| Line 233: | Line 233: | ||
</div> | </div> | ||
<p id="fig-text"><i><b>Figure 1:</b>SDS-page and western blot. Expression of Tyrosine ammonia lyase with constitutive promotors. The negative control is empty, showing that there is no natural protein in e-coli with the same attributes.</i><br><br></p> | <p id="fig-text"><i><b>Figure 1:</b>SDS-page and western blot. Expression of Tyrosine ammonia lyase with constitutive promotors. The negative control is empty, showing that there is no natural protein in e-coli with the same attributes.</i><br><br></p> | ||
| + | |||
| + | <img class="results_pic_wblot_pc" src="https://static.igem.org/mediawiki/igem.org/d/da/Uppsala_pic_coumaric_Spectrophotometry.png"> | ||
| + | <p id="fig-text"><i><b>Figure 2:</b>Absorbance spectra of extracts collected from bacterial cultures of strain pSB3K3-CP8-TAL. Samples were collected 21 h and 48 h after 30 °C incubation. Negative control is an extract from a strain with no TAL gene on the transformed plasmid. The positive control is an extract on a culture of the same strain as the negative control but with added p-coumaric acid to a concentration of 500 µM before extraction.</i> | ||
<table> | <table> | ||
Revision as of 21:17, 1 October 2013
Results and achievement"
Achievement
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 d5halpha and e-coli nissle. For detailed information about the characterisation methods, see the protocol section.
2. TAL with Cp8 promotor
3. TAL with J23110 promotor
4. Negative control
Figure 1:SDS-page and western blot. Expression of Tyrosine ammonia lyase with constitutive promotors. 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 of strain pSB3K3-CP8-TAL. Samples were collected 21 h and 48 h after 30 °C incubation. Negative control is an extract from a strain with no TAL gene on the transformed plasmid. The positive control is an extract on a culture of the same strain as the negative control but with added p-coumaric acid to a concentration of 500 µM before extraction.
 | 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 ~9 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 9 minutes. |
