Team:ITESM-CEM/Interlab
From 2014.igem.org
(12 intermediate revisions not shown) | |||
Line 97: | Line 97: | ||
GFP has long been used as a reporter of patterns of gene expression in both prokaryotes, were it is useful for characterization of promoters, enhancers and terminators; and in eukaryotes, were tissue-specific or time-specific gene expression can be traced (2). The basis of this procedure is the usage of GFP’s fluorescence as a reporter of activity of promoters and enhancers; the relative fluorescence of cells at different experimental conditions can be compared with statistical techniques, and so the efficiency of the parts can be tested. | GFP has long been used as a reporter of patterns of gene expression in both prokaryotes, were it is useful for characterization of promoters, enhancers and terminators; and in eukaryotes, were tissue-specific or time-specific gene expression can be traced (2). The basis of this procedure is the usage of GFP’s fluorescence as a reporter of activity of promoters and enhancers; the relative fluorescence of cells at different experimental conditions can be compared with statistical techniques, and so the efficiency of the parts can be tested. | ||
</p> | </p> | ||
- | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | + | <gotop><a href="#top"><b>Back to top ↑</b></a></gotop><br><br> |
<a name="Protocols"> <u><h2>Protocols</h2></u> </a> | <a name="Protocols"> <u><h2>Protocols</h2></u> </a> | ||
Line 125: | Line 125: | ||
To ligate, 2 μl of water for molecular biology, 2 μl of NEB® T4 Ligase buffer, 8 μl of the first digestion product (BBa_K823005), 4 μl of the second digestion product (BBa_E0240), 5 μl of the third digestion product (psB1C3 backbone), and 1 μl of NEB® T4 Ligase were added to a 0.5 ml PCR tube. The mixture was incubated at 16°C for 24 hours, then inactivated for 10 minutes at 65°C and finally stored at -20°C. | To ligate, 2 μl of water for molecular biology, 2 μl of NEB® T4 Ligase buffer, 8 μl of the first digestion product (BBa_K823005), 4 μl of the second digestion product (BBa_E0240), 5 μl of the third digestion product (psB1C3 backbone), and 1 μl of NEB® T4 Ligase were added to a 0.5 ml PCR tube. The mixture was incubated at 16°C for 24 hours, then inactivated for 10 minutes at 65°C and finally stored at -20°C. | ||
</p> | </p> | ||
- | <br>< | + | <br> |
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/7/79/10702886_354506261379425_754230058_o.jpg" align="right" width="620" height="700" hspace="10" BORDER=10> | ||
<h4>Fluorescence Measurement Protocol</h4> | <h4>Fluorescence Measurement Protocol</h4> | ||
Line 134: | Line 136: | ||
<br> | <br> | ||
</p> | </p> | ||
- | <img src="https://static.igem.org/mediawiki/2014/c/c7/Rsz_flourskan.jpg" align="center" width="400" height="225" hspace="10" BORDER=10> | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/c/c7/Rsz_flourskan.jpg" align="center" width="400" height="225" hspace="10" BORDER=10></p> |
- | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | + | <br><br><gotop><a href="#top"><b>Back to top ↑</b></a></gotop><br><br> |
Line 146: | Line 148: | ||
In order to compare fragment sizes, individual BioBricks were also digested. Promoter BBa_K823012, contained in backbone psB1C3 was digested using EcoRI and SpeI; and mutant GFP gene, BBa_E0240 (also contained in psB1C3 plasmid backbone) was digested with XbaI and PstI.<br> | In order to compare fragment sizes, individual BioBricks were also digested. Promoter BBa_K823012, contained in backbone psB1C3 was digested using EcoRI and SpeI; and mutant GFP gene, BBa_E0240 (also contained in psB1C3 plasmid backbone) was digested with XbaI and PstI.<br> | ||
- | <img src="https://static.igem.org/mediawiki/2014/6/6d/Interlab_Results_%282%29-3.jpg" width="400" height="400" hspace="20" BORDER=10><br> | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/6/6d/Interlab_Results_%282%29-3.jpg" width="400" height="400" hspace="20" BORDER=10><br></p> |
- | <pie><b>Figure 1.</b> Results of DNA electrophoresis in 0.8% agarose gel for each BioBrick, and assembled device. Four 1 kb plus markers are included, and each lane is labelled with the BioBrick or device’s name. Device 1 is represented by the set of lanes labelled BBa_I20260.</pie> | + | <p><pie><b>Figure 1.</b> Results of DNA electrophoresis in 0.8% agarose gel for each BioBrick, and assembled device. Four 1 kb plus markers are included, and each lane is labelled with the BioBrick or device’s name. Device 1 is represented by the set of lanes labelled BBa_I20260.</pie></p> |
- | < | + | |
- | Restriction analyses for each individual BioBrick (figure 1) appear to be correct, excepting a low number of upper bands generated by ligation of infrequent plasmid isoforms. However, electrophoresis results for the assembled devices appear blurred in most cases; since this result is generalized for all DNA samples obtained via alkaline extraction (including lanes for BBa_I20260), it is thought that an excessive contamination was obtained when performing plasmid extraction, as revealed by wide and dark RNA bands at the lower area of the gels, as well as widespread DNA degradation. Even though it is not possible to analyse the structure of the assembled devices as efficiently as was needed, it is clear that this is not a prove of the existence of errors in transformation protocols, since device 1 fluoresces, and still cannot be seen at the restriction assay. | + | <p style="text-align: justify; text-justify: inter-word;">Restriction analyses for each individual BioBrick (figure 1) appear to be correct, excepting a low number of upper bands generated by ligation of infrequent plasmid isoforms. However, electrophoresis results for the assembled devices appear blurred in most cases; since this result is generalized for all DNA samples obtained via alkaline extraction (including lanes for BBa_I20260), it is thought that an excessive contamination was obtained when performing plasmid extraction, as revealed by wide and dark RNA bands at the lower area of the gels, as well as widespread DNA degradation. Even though it is not possible to analyse the structure of the assembled devices as efficiently as was needed, it is clear that this is not a prove of the existence of errors in transformation protocols, since device 1 fluoresces, and still cannot be seen at the restriction assay. |
Device 3 is the only exception to these observations, since its control lane presents a band of the appropriate size; however, the restriction lane does not correspond with the predicted fragment sizes.<br><br> | Device 3 is the only exception to these observations, since its control lane presents a band of the appropriate size; however, the restriction lane does not correspond with the predicted fragment sizes.<br><br> | ||
Given the previous analysis, the only possible explanation for the lack of fluorescence of devices 2 and 3 is an error in either the sequence or the cloning procedures for promoters in plasmid psB1C3; or an unexpected pattern of BioBrick ligation. It is then concluded that no efficient activity can be characterized for Promoter BBa_K823012 or for any of the BioBricks assembled with psB1C3 plasmid backbone. | Given the previous analysis, the only possible explanation for the lack of fluorescence of devices 2 and 3 is an error in either the sequence or the cloning procedures for promoters in plasmid psB1C3; or an unexpected pattern of BioBrick ligation. It is then concluded that no efficient activity can be characterized for Promoter BBa_K823012 or for any of the BioBricks assembled with psB1C3 plasmid backbone. | ||
</p> | </p> | ||
- | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | + | <gotop><a href="#top"><b>Back to top ↑</b></a></gotop><br><br> |
Line 161: | Line 162: | ||
<p style="text-align: justify; text-justify: inter-word;"> | <p style="text-align: justify; text-justify: inter-word;"> | ||
Relative Fluorescence Units (RFU) for each bacterial strain, as determined by triplicate with the fluorometer for each overnight culture and 2-hour subculture sample are reported at Appendix A. Further, a statistical mean, as well as a value of standard deviation, was calculated for each triplicate of measurements; this data is also summarized in Appendix A. | Relative Fluorescence Units (RFU) for each bacterial strain, as determined by triplicate with the fluorometer for each overnight culture and 2-hour subculture sample are reported at Appendix A. Further, a statistical mean, as well as a value of standard deviation, was calculated for each triplicate of measurements; this data is also summarized in Appendix A. | ||
- | The relative fluorescence of both negative controls (DH5α and Top 10 strains) with respect to the optical density is presented in dispersion plots (figure 2). A smooth negative association was found for both sets of samples. The negative slope of the curve indicates that the biomass may absorb some of the basal fluorescence.<br><br> | + | The relative fluorescence of both negative controls (DH5α and Top 10 strains) with respect to the optical density is presented in dispersion plots (figure 2). A smooth negative association was found for both sets of samples. The negative slope of the curve indicates that the biomass may absorb some of the basal fluorescence.<br><br></p> |
- | <img src="https://static.igem.org/mediawiki/2014/f/f9/Interlab_Results_%282%29-2.jpg" align="middle" hspace="10" BORDER=10><br> | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/f/f9/Interlab_Results_%282%29-2.jpg" align="middle" hspace="10" BORDER=10><br> |
<b>Figure 2.</b> Mean Relative Fluorescence plot for negative control samples taken from overnight cultures at increasing optical densities. All correlation coefficients are shown. | <b>Figure 2.</b> Mean Relative Fluorescence plot for negative control samples taken from overnight cultures at increasing optical densities. All correlation coefficients are shown. | ||
</p> | </p> | ||
- | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | + | <gotop><a href="#top"><b>Back to top ↑</b></a></gotop><br><br> |
Line 173: | Line 174: | ||
<p style="text-align: justify; text-justify: inter-word;"> | <p style="text-align: justify; text-justify: inter-word;"> | ||
The values of relative fluorescence for the DH5α negative control were then subtracted from the lectures obtained when analysing the modified bacterial strains. Dispersion plots for each genetic device built this way are presented in figure 3. An analysis of correlation reveals a strong positive association between relative fluorescence and OD600 in E. coli transformed with BioBrick BBa_I20260 (device 1) for both samples (overnight culture and 2-hour subculture). However, there appears to be no increase in fluorescence on the plots for BioBrick BBa_K823005 + BBa_E0240 (device 2) and BBa_K823012 + BBa_E0240 (device3). | The values of relative fluorescence for the DH5α negative control were then subtracted from the lectures obtained when analysing the modified bacterial strains. Dispersion plots for each genetic device built this way are presented in figure 3. An analysis of correlation reveals a strong positive association between relative fluorescence and OD600 in E. coli transformed with BioBrick BBa_I20260 (device 1) for both samples (overnight culture and 2-hour subculture). However, there appears to be no increase in fluorescence on the plots for BioBrick BBa_K823005 + BBa_E0240 (device 2) and BBa_K823012 + BBa_E0240 (device3). | ||
- | Finally, the curves of relative fluorescence for overnight culture and 2-hour subculture samples of bacteria expressing BioBrick BBa_I20260 are superposed in a single plot (figure 4). Correlation (R2) is kept almost constant; but there is a clear increase in the slope of the line when comparing the 37°C, 2-hour subculture with the regular overnight culture stored at 4°C. <br><br> | + | Finally, the curves of relative fluorescence for overnight culture and 2-hour subculture samples of bacteria expressing BioBrick BBa_I20260 are superposed in a single plot (figure 4). Correlation (R2) is kept almost constant; but there is a clear increase in the slope of the line when comparing the 37°C, 2-hour subculture with the regular overnight culture stored at 4°C. <br><br></p> |
- | <img src="https://static.igem.org/mediawiki/2014/9/90/Interlab_Results_%282%29-5.jpg" align="middle" hspace="10" BORDER=10><br> | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/9/90/Interlab_Results_%282%29-5.jpg" align="middle" hspace="10" BORDER=10><br> |
- | <b>Figure 3.</b> Mean Relative Fluorescence plot for E. coli DH5α transformed with each of the three genetic devices, samples taken from overnight cultures at increasing optical densities. The basal levels of fluorescence were subtracted. All correlation coefficients are shown.<br><br> | + | <b>Figure 3.</b> Mean Relative Fluorescence plot for E. coli DH5α transformed with each of the three genetic devices, samples taken from overnight cultures at increasing optical densities. The basal levels of fluorescence were subtracted. All correlation coefficients are shown.<br><br></p> |
- | <img src="https://static.igem.org/mediawiki/2014/5/56/Interlab_Results_%282%29-8.jpg" align="middle" hspace="10" BORDER=10><br> | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/5/56/Interlab_Results_%282%29-8.jpg" align="middle" hspace="10" BORDER=10><br> |
<b>Figure 4.</b> Comparative plot of Mean Relative Fluorescence for genetically modified E. coli DH5α strain, transformed with BioBrick BBa_I20260, at increasing optical densities. The upper curve belongs to a sample taken of the 2-hour subculture at 37°C in LB media; while the lower one was obtained directly from an overnight culture stored at 4°C. The basal levels of fluorescence emission were subtracted. All correlation coefficients are shown. | <b>Figure 4.</b> Comparative plot of Mean Relative Fluorescence for genetically modified E. coli DH5α strain, transformed with BioBrick BBa_I20260, at increasing optical densities. The upper curve belongs to a sample taken of the 2-hour subculture at 37°C in LB media; while the lower one was obtained directly from an overnight culture stored at 4°C. The basal levels of fluorescence emission were subtracted. All correlation coefficients are shown. | ||
</p> | </p> | ||
- | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | + | <gotop><a href="#top"><b>Back to top ↑</b></a></gotop><br><br> |
<a name="GFP1"><u><h2>Device 1 (BBa_I20260)</h2></u></a> | <a name="GFP1"><u><h2>Device 1 (BBa_I20260)</h2></u></a> | ||
Line 189: | Line 190: | ||
</p> | </p> | ||
- | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | + | <gotop><a href="#top"><b>Back to top ↑</b></a></gotop><br><br> |
<a name="GFP2"><u><h2>Device 2 & 3 (BBa_K823005+BBa_E0240, BBa_K823012+BBa_E0240)</h2></u></a> | <a name="GFP2"><u><h2>Device 2 & 3 (BBa_K823005+BBa_E0240, BBa_K823012+BBa_E0240)</h2></u></a> | ||
Line 196: | Line 197: | ||
</p> | </p> | ||
- | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | + | <gotop><a href="#top"><b>Back to top ↑</b></a></gotop><br><br> |
<a name="Conclusion"><u><h2>Conclusions</h2></u></a> | <a name="Conclusion"><u><h2>Conclusions</h2></u></a> | ||
Line 205: | Line 206: | ||
</p> | </p> | ||
- | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | + | <gotop><a href="#top"><b>Back to top ↑<b></a></gotop><br><br> |
<a name="Annex"><u><h2>Annexes</h2></u></a> | <a name="Annex"><u><h2>Annexes</h2></u></a> | ||
<p style="text-align: justify; text-justify: inter-word;"> | <p style="text-align: justify; text-justify: inter-word;"> | ||
- | Measurements taken for 150 μl samples of overnight bacterial cultures after 2 hours of incubation at 4°C | + | Measurements taken for 150 μl samples of overnight bacterial cultures after 2 hours of incubation at 4°C</p> |
- | <img src="https://static.igem.org/mediawiki/2014/7/79/Table1_%282%29-8.jpg" width="600" height="550" align="middle" hspace="10" BORDER=10><br><br> | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/7/79/Table1_%282%29-8.jpg" width="600" height="550" align="middle" hspace="10" BORDER=10><br><br></p> |
- | Measurements taken for 150 μl samples of overnight cultures of negative controls, and E. coli DH5α transformed with BioBrick BBa_I20260. Samples were subcultured for 2 hours at 37°C and constant stirring.<br> | + | <p style="text-align: justify; text-justify: inter-word;">Measurements taken for 150 μl samples of overnight cultures of negative controls, and E. coli DH5α transformed with BioBrick BBa_I20260. Samples were subcultured for 2 hours at 37°C and constant stirring.<br></p> |
- | <img src="https://static.igem.org/mediawiki/2014/b/b7/Table2_%282%29-9.jpg" width="500" height="500" align="middle" hspace="10" BORDER=10><br><br> | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/b/b7/Table2_%282%29-9.jpg" width="500" height="500" align="middle" hspace="10" BORDER=10><br><br></p> |
- | Statistical means and standard deviations calculated for each triplicate of measurements of relative fluorescence, using samples taken from overnight cultures stored for 2 hours at 4°C. | + | <p style="text-align: justify; text-justify: inter-word;">Statistical means and standard deviations calculated for each triplicate of measurements of relative fluorescence, using samples taken from overnight cultures stored for 2 hours at 4°C.</p> |
- | <img src="https://static.igem.org/mediawiki/2014/9/9b/Table3_%282%29-10.jpg" width="700" height="250" align="middle" hspace="10" BORDER=10><br><br> | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/9/9b/Table3_%282%29-10.jpg" width="700" height="250" align="middle" hspace="10" BORDER=10><br><br></p> |
- | Statistical means and standard deviations calculated for each triplicate of measurements of relative fluorescence, using samples taken from E. coli DH5α transformed with BioBrick BBa_I20260, subcultured for 2 hours at 37°C and constant stirring.<br> | + | <p style="text-align: justify; text-justify: inter-word;">Statistical means and standard deviations calculated for each triplicate of measurements of relative fluorescence, using samples taken from E. coli DH5α transformed with BioBrick BBa_I20260, subcultured for 2 hours at 37°C and constant stirring.<br></p> |
- | <img src="https://static.igem.org/mediawiki/2014/2/29/Table4_%282%29-10.jpg" width="380" height="350" align="middle" hspace="10" BORDER=10> | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/2/29/Table4_%282%29-10.jpg" width="380" height="350" align="middle" hspace="10" BORDER=10> |
</p> | </p> | ||
- | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | + | <gotop><a href="#top"><b>Back to top ↑</b></a></gotop><br><br> |
<u><h2>References</h2></u> | <u><h2>References</h2></u> |
Latest revision as of 19:14, 15 October 2014
ITESM-CEM | Enzy7-K me |
|
![]() |
|||
Interlab
|
![]() |
||||
| ![]() |
||||
![]() |
|||||
![]() |
|||||
![]() |
|||||
|
![]() |
||||
![]() |
![]() |
![]() |
![]() |
![]() |
|