Team:Lethbridge/results
From 2013.igem.org
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<center><image src="https://static.igem.org/mediawiki/2013/6/6a/ULeth2013_PK401-overexpression.png"; width="450px"; height="250px" /></center> | <center><image src="https://static.igem.org/mediawiki/2013/6/6a/ULeth2013_PK401-overexpression.png"; width="450px"; height="250px" /></center> | ||
- | <p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p> | + | <p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br> |
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p> | <p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p> | ||
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- | <p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p> | + | <p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br> |
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p> | <p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p> | ||
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<center><image src="https://static.igem.org/mediawiki/2013/b/b3/ULeth2013_PK401-CFPexcitation.png"; width="450px"; height="250px" /></center> | <center><image src="https://static.igem.org/mediawiki/2013/b/b3/ULeth2013_PK401-CFPexcitation.png"; width="450px"; height="250px" /></center> | ||
- | <p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p> | + | <p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p><br> |
<p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p> | <p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p> | ||
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<center><image src="https://static.igem.org/mediawiki/2013/3/35/ULeth2013_PK401-YFPexcitation.png"; width="450px"; height="250px" /></center> | <center><image src="https://static.igem.org/mediawiki/2013/3/35/ULeth2013_PK401-YFPexcitation.png"; width="450px"; height="250px" /></center> | ||
- | <p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p> | + | <p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p><br> |
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p> | <p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p> | ||
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<center><image src="https://static.igem.org/mediawiki/2013/c/c3/ULeth2013_PK401-FRET.png"; width="450px"; height="250px" /></center> | <center><image src="https://static.igem.org/mediawiki/2013/c/c3/ULeth2013_PK401-FRET.png"; width="450px"; height="250px" /></center> | ||
- | <p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p> | + | <p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br> |
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p> | <p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p> |
Revision as of 19:00, 27 September 2013
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