Difference between revisions of "Team:SDU-Denmark/Tour52"
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− | <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=" | + | <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 37oC 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 37oC overnight. Only cells that were co-transformed with pSB1C3-T18-Zip + pSB1K3-T25-Zip got blue, while the others remained white. This indicates that beta-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> | <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 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. | ||
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− | <a class="popupImg alignRight" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2015/0/02/SDU2015_fluorescenceimagev2.png" title=" | + | <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> | <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 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). |
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Revision as of 00:59, 19 September 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. This is system is based on the reconstitution of the adenylate cyclase and allows detection of protein-protein interaction. In this page we validate the function of the two-hybrid system.
To validate that 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 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. 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. 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 deficiency in the gene encoding the 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. 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. However, 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 out on LB/X-gal plates with appropriate 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 showed complementation between T18 and T25 when the leucine zipper was fused to both of the domains. These results prove that our constructs function as expected, and that it 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 should be observed instead of blue. The transformations should however be plated out on LB plates without X-gal.
We wanted to carry out a screening for peptide aptamers, but due to late deliverance of our nucleotide library we did not reach to accomplish this.
Green Fluorescent Protein was fused to the two components of the two-hybrid system, T18 and T25. This allows us to detect expression of these two components that are both under control of a 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 under the lac promoter, and that proteins fused to these two constructs will fold into the correct structure.
The two constructs pSB1C3-T18-GFP and pSB1C3-T25-GFP were transformed into the competent E. coli K12-strain TOP10. The fluorescence microscopy images (fig. 2) confirm the presence of green fluorescence. This means that both T18 and T25 are expressed under the lac promoter, and that proteins fused to these two constructs will fold into the correct structure.