Difference between revisions of "Team:SDU-Denmark/Tour52"
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+ | <p> <i> "Alone we can do so little; together we can do so much." - <b>Helen Keller</b></i></p> | ||
+ | <h1 align="center">Two-Hybrid System</h1> | ||
+ | <div class="thumb tright"> | ||
+ | <div class="thumbinner" style="width:260px;height:395px;"> | ||
+ | <a class="popupImg alignRight" style="width:255px" target="_blank" href="https://static.igem.org/mediawiki/2015/d/de/Two-hybrid-system1-SDU_Denmark.png" title="Plate streaking of transformed BTH101 on LB/X-gal plates. Competent BTH101 cells were co-transformed with the plasmids pSB1C3-T18 + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25 and pSB1C3-T18-Zip + pSB1K3-T25-Zip. Transformations were plated out on LB plates containing 40 µg/ml x-gal, 30 µg/ml kanamycin and 25 µg/ml chloramphenicol and incubated at 37<sup>o</sup>C overnight. Colonies from successful transformation were picked out and streaked out on the same LB/x-gal plate in a matrix as shown above. Cells were again incubated at 37<sup>o</sup>C overnight. Only cells that were co-transformed with pSB1C3-T18-Zip + pSB1K3-T25-Zip got blue, while the others remained white. This indicates that β-Galactosidase was expressed in these cells and that the adenylate cyclase activity was reconstituted. This confirms the interaction of the leucine zippers, and validates the function of the bacterial two-hybrid system. " title="Streaks of BTH101"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/d/de/Two-hybrid-system1-SDU_Denmark.png" style="width:255px"/> </a> | ||
+ | <div class="thumbcaption">Figure 1: Plate streaking of transformed BTH101 on LB/X-gal plates, containing pSB1C3-T18 + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25 and pSB1C3-T18-Zip + pSB1K3-T25-Zip. | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
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<p> | <p> | ||
− | < | + | <span class="intro">In order to</span> make a great screening for peptide aptamers you need a well-functioning screening system. Our screening system is the bacterial two-hybrid system, which is based on the reconstitution of the adenylate cyclase and enables detection of protein-protein interaction. Here we validate the function of our bacterial two-hybrid system. |
</p> | </p> | ||
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<p> | <p> | ||
− | < | + | <span class="intro">To validate</span> whether our T18 and T25 domain constructs in fact can be used to study protein-protein interactions, we made a control experiment, where the leucine zipper (Zip) region from the GCN4 yeast (<i>Saccharomyces cerevisiae</i>) protein was fused to the T18 and T25 domains (T18-Zip+T25-Zip). Leucine zippers are known to interact by forming homodimers and if the system indeed works, their interaction will lead to functional complementation between the T18 and T25 domains. This leads to the synthesis of cAMP, and thus by using a cAMP-induced reporter system, one can observe whether or not there is an interaction. |
+ | </p> | ||
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− | < | + | <p> |
− | < | + | <span class="intro">When working</span> with the bacterial two-hybrid system, it is necessary to use a strain with a non-functioning or absent adenylate cyclase, <i>cyaA</i>. For this purpose we constructed the <i>Escherichia coli</i> K12-strain, MG1655 Δ<i>cyaA</i>. We also used the <i>cyaA</i>-deficient <i>E. coli</i> K12-strain BTH101 (MC1061-derived). This was for the purpose to exploit the <i>lacZ</i>-derived reporter system encoded in this strains chromosome. The <i>lacZ</i> gene encodes a β-Galactosidase which is positively controlled by cAMP. Our goal was to use the RFP reporter system in the MG1655 Δ<i>cyaA</i>-strain. Due to difficulties with RFP reporter system, this control experiment was carried out in the BTH101-strain. |
− | < | + | </p> |
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− | < | + | |
− | </ | + | |
− | </ | + | |
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− | < | + | <span class="tooltipLink">Four different combinations</span><span class="tooltip"> |
− | + | <span class="tooltipHeader">Four different combinations</span> | |
− | < | + | <p> |
+ | <ul> | ||
<li>pSB1C3-T18+pSB1K3-T25</li> | <li>pSB1C3-T18+pSB1K3-T25</li> | ||
<li>pSB1C3-T18+pSB1K3-T25-Zip</li> | <li>pSB1C3-T18+pSB1K3-T25-Zip</li> | ||
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<li>pSB1C3-T18-Zip+pSB1K3-T25-Zip</li> | <li>pSB1C3-T18-Zip+pSB1K3-T25-Zip</li> | ||
</ul> | </ul> | ||
+ | </p> | ||
− | < | + | <p> |
+ | These transformations were plated on LB/X-gal plates with appropriately selective antibiotics and 40 µg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). | ||
+ | </p> | ||
+ | </span> | ||
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− | + | of plasmids were sequentially co-transformed into the BTH101-strain. | |
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</p> | </p> | ||
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<p> | <p> | ||
− | < | + | <span class="intro">As expected</span>, the results only displayed complementation between T18 and T25 when the leucine zipper was fused to both of the domains. These results infers that our constructs function as expected, and that they can be used to detect protein-protein interactions. This indicates that the system can be used to screen for peptide aptamers. |
− | + | If we wanted to use the RFP reporter system instead, the same experiment could be conducted and red colonies would be observed instead of blue. The transformations should, however, be plated on LB plates without X-gal. | |
− | + | </p> | |
<p> | <p> | ||
− | < | + | <span class="intro">We wanted to carry out a screening</span> for peptide aptamers, but due to delayed deliverance of our nucleotide library and problems inserting it into our scaffold, we did not manage to accomplish this in the given time frame. |
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</p> | </p> | ||
+ | <div class="thumb tright"> | ||
+ | <div class="thumbinner" style="width:505px;height:200px;"> | ||
+ | <a class="popupImg alignRight" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2015/0/02/SDU2015_fluorescenceimagev2.png" title="Figure 2: A culture of <i>Escherichia coli</i> Top10 cells containing the plasmids: pSB1C3-T25-GFP, pSB1C3-T18-GFP and pSB1C3-T18 were grown overnight. The overnight culture was diluted 1:100 and grown into an exponential phase, where the cells were spinned down and washed in phospate-buffered saline (PBS) and then resuspended in PBS. The cells were fixated on a glass slide with an isotonic agarose gel. Fluorescence microscopy images were acquired by exciting the proteins with a blue laser at 475 nm. From the images acquired it can be concluded that both pSB1C3-T25-GFP (A) and pSB1C3-T18-GFP (B), as expected, showed green fluorescence, while pSB1C3-T18 (C) did not." title="Figure 2: A culture of <i>Escherichia coli</i> Top10 cells containing the plasmids: pSB1C3-T25-GFP, pSB1C3-T18-GFP and pSB1C3-T18 were grown overnight. The overnight culture was diluted 1:100 and grown into an exponential phase, where the cells were spinned down and washed in phospate-buffered saline (PBS) and then resuspended in PBS. The cells were fixated on a glass slide with an isotonic agarose gel. Fluorescence microscopy images were acquired by exciting the proteins with a blue laser at 475 nm. From the images acquired it can be concluded that both pSB1C3-T25-GFP (A) and pSB1C3-T18-GFP (B), as expected, show green fluorescence, while pSB1C3-T18 (C) did not."> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/c/c8/SDU2015_fluorescenceimagev2lille.png" style="width:500px"/> </a> | ||
+ | <div class="thumbcaption">Figure 2: Fluorescence microscopy images: (A) pSB1C3-T25-GFP, (B) pSB1C3-T18-GFP and (C) pSB1C3-T18 transformed into Top10 (<i>E. coli</i> K12-strain). | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
− | + | <p> | |
+ | <span class="intro">Green Fluorescent Protein</span> was fused to the two components of the two-hybrid system, T18 and T25. This allowed us to detect expression of the two components that are both controlled by the lac promoter. Additionally, the presence of green fluorescence will verify that proteins can be fused to T18 and T25, and still fold into the correct structure. | ||
+ | The two constructs pSB1C3-T18-GFP and pSB1C3-T25-GFP were transformed into the <i>E. coli</i> K12-strain Top10. The following fluorescence microscopy images confirm the presence of green fluorescence. This means that both T18 and T25 are expressed from the lac promoter, and that proteins fused to these two constructs can fold into their native structure. | ||
</p> | </p> | ||
+ | |||
+ | <p> | ||
+ | <span class="intro">The two constructs</span>, pSB1C3-T18-GFP and pSB1C3-T25-GFP, were transformed into the competent <i>E. coli</i> K12-strain, Top10. Fluorescence microscopy imaging (fig. 2) confirms the presence of green fluorescence, thus both T18 and T25 are expressed by the lac promoter, and that GFP fused to the two constructs can fold into its correct structure. This indicates that proteins can be fused to T18 and T25, without affecting their 3D-structure. | ||
+ | </p> | ||
</html> | </html> | ||
{{:Team:SDU-Denmark/core/footer}} | {{:Team:SDU-Denmark/core/footer}} |
Latest revision as of 16:23, 4 October 2015
"Alone we can do so little; together we can do so much." - Helen Keller
Two-Hybrid System
In order to make a great screening for peptide aptamers you need a well-functioning screening system. Our screening system is the bacterial two-hybrid system, which is based on the reconstitution of the adenylate cyclase and enables detection of protein-protein interaction. Here we validate the function of our bacterial two-hybrid system.
To validate whether our T18 and T25 domain constructs in fact can be used to study protein-protein interactions, we made a control experiment, where the leucine zipper (Zip) region from the GCN4 yeast (Saccharomyces cerevisiae) protein was fused to the T18 and T25 domains (T18-Zip+T25-Zip). Leucine zippers are known to interact by forming homodimers and if the system indeed works, their interaction will lead to functional complementation between the T18 and T25 domains. This leads to the synthesis of cAMP, and thus by using a cAMP-induced reporter system, one can observe whether or not there is an interaction.
When working with the bacterial two-hybrid system, it is necessary to use a strain with a non-functioning or absent adenylate cyclase, cyaA. For this purpose we constructed the Escherichia coli K12-strain, MG1655 ΔcyaA. We also used the cyaA-deficient E. coli K12-strain BTH101 (MC1061-derived). This was for the purpose to exploit the lacZ-derived reporter system encoded in this strains chromosome. The lacZ gene encodes a β-Galactosidase which is positively controlled by cAMP. Our goal was to use the RFP reporter system in the MG1655 ΔcyaA-strain. Due to difficulties with RFP reporter system, this control experiment was carried out in the BTH101-strain.
Four different combinations Four different combinations
- pSB1C3-T18+pSB1K3-T25
- pSB1C3-T18+pSB1K3-T25-Zip
- pSB1C3-T18-Zip+pSB1K3-T25
- pSB1C3-T18-Zip+pSB1K3-T25-Zip
These transformations were plated on LB/X-gal plates with appropriately selective antibiotics and 40 µg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside).
of plasmids were sequentially co-transformed into the BTH101-strain.As expected, the results only displayed complementation between T18 and T25 when the leucine zipper was fused to both of the domains. These results infers that our constructs function as expected, and that they can be used to detect protein-protein interactions. This indicates that the system can be used to screen for peptide aptamers. If we wanted to use the RFP reporter system instead, the same experiment could be conducted and red colonies would be observed instead of blue. The transformations should, however, be plated on LB plates without X-gal.
We wanted to carry out a screening for peptide aptamers, but due to delayed deliverance of our nucleotide library and problems inserting it into our scaffold, we did not manage to accomplish this in the given time frame.
Green Fluorescent Protein was fused to the two components of the two-hybrid system, T18 and T25. This allowed us to detect expression of the two components that are both controlled by the lac promoter. Additionally, the presence of green fluorescence will verify that proteins can be fused to T18 and T25, and still fold into the correct structure. The two constructs pSB1C3-T18-GFP and pSB1C3-T25-GFP were transformed into the E. coli K12-strain Top10. The following fluorescence microscopy images confirm the presence of green fluorescence. This means that both T18 and T25 are expressed from the lac promoter, and that proteins fused to these two constructs can fold into their native structure.
The two constructs, pSB1C3-T18-GFP and pSB1C3-T25-GFP, were transformed into the competent E. coli K12-strain, Top10. Fluorescence microscopy imaging (fig. 2) confirms the presence of green fluorescence, thus both T18 and T25 are expressed by the lac promoter, and that GFP fused to the two constructs can fold into its correct structure. This indicates that proteins can be fused to T18 and T25, without affecting their 3D-structure.