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Team:Technion/Project/RS
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| - | == | + | ==What is a Riboswitch?== |
| + | [[File:RS_figure1.jpg|300px|thumb|right|<strong><em>Figure 1:</em></strong><em> predicted mechanism of 12.1 Riboswitch. Adopted from {1}.</em>]] | ||
| + | <p align="left">Riboswitches are regulatory elements and can control translation of genes. They consist of a short mRNA sequence that can fold to create a structure. This structure can be changed in the presence of a specific ligand. The regulatory function is achieved if the mRNA structural change can determine if a gene will be translated. In bacteria this can be done by placing the riboswitch before a RBS and ensuring that it will block the ribosome from the RBS, and that after the ligand is added the structural change will allow the ribosome access to the RBS. It is important to note that the function of the RS can be affected by the coding region downstream to it {1,2}. | ||
| + | <br clear="all" /></p> | ||
| - | ==The assembly PCR | + | ==Fusion PCR== |
| + | <p align="left">The technique is also known as [http://openwetware.org/wiki/Assembly_pcr assembly PCR]. This technique allows fusing together any two DNA pieces in a precise way, and without having to rely on restriction sites. This is accomplished by careful planning of primes so that for each DNA piece there are 2 primers: in the fused area the primer has a complementary area for the DNA piece and also a complementary area for the second DNA piece; normal primer for the non-fused end {3}.<br /> | ||
| + | The first step is to elongate each of the DNA pieces so that each will contain a complementary region for the other DNA piece. This is done by normal PCR {3}.<br /> | ||
| + | </p> | ||
| + | [[File:fusion_pcr1.jpg|600px|center]] | ||
| + | <p align="left">The second step is to elongate both DNA pieces in the same PCR reaction using only the outer primers. This way the overlapping region of the DNA pieces will act as a third primer, resulting in fused product that contains both DNA pieces {3}. <br /> | ||
| + | </p> | ||
| + | [[File:fusion_pcr2.jpg|500px|center]] | ||
| - | ==The planned | + | ==General design== |
| + | [[File:RS_figure3.jpg|300px|thumb|right|<strong><em>Figure 3:</em></strong> <em>summary of our fusion PCR strategy.</em>]] | ||
| + | <p>We used a synthetic theophylline riboswitch, clone 12.1, that was created by S.A Lynch and J.P Gallivan{1}.<br /> | ||
| + | We chose to use this kind of regulatory element for the following reasons:</p> | ||
| + | <ol> | ||
| + | <li><span dir="ltr"> </span>In our project we are using several different regulatory elements and we need them to work together with little or none crosstalk. Most of the regulatory elements are inducible promoters. We had a hard time finding more inducible promoters that are well defined and don’t crosstalk with the rest of our promoters. So we looked for another kind of regulatory element.</li> | ||
| + | <li><span dir="ltr"> </span>Riboswitches were discovered only in 2002, and the synthetic ones were created not long after.</li> | ||
| + | <li><span dir="ltr"> </span>Riboswitches can be useful to future iGEM teams and the rest of the synthetic biology community as an additional and unique regulatory element. </li> | ||
| + | </ol> | ||
| + | [[File:RS_figure2.jpg|300px|thumb|left|<strong><em>Figure 2:</em></strong><em> Riboswitch with RNA polymerase or mCherry cassette.</em>]] | ||
| + | <p align="left">In our project we use Riboswitches with a reporter gene (mCherry) and RNA polymerases: T7, T7*, SP6, T7*(T3),T7*( K1F), T7*(N4) (later notated as T3, K1F and N4){4}. <br /> | ||
| + | In order to combine the riboswitch with the different genes we use fusion PCR. We use it because in order for the riboswitch to work the start codon of the gene should be adjacent and downstream to the RBS. This is due to the start codon part in the specific riboswitch structure. <br /> | ||
| + | The cassettes that are used in the plasmids are shown in <strong><em>Figure 2.</em></strong><br /> | ||
| + | </p> | ||
| + | <p>The Riboswitch is very short (74 bp including the RBS) and it could be a problem with PCR, so we’ve extracted it with 430 bp upstream (part of the pUC19 plasmid) and the long Riboswitch is then 504 bp long. Moreover, the added part from pUC19 contains an MCS (which contains an EcoRI site) and a tac promoter. By adding a PstI site to the end of the RNAP polymerases or mCherry (by carefully planning the primer) we could clone the fusion product into pSB1C3. <br /> | ||
| + | The summary of our fusion PCR strategy is presented in <strong><em>Figure 3</em></strong>. | ||
| + | <br clear="all" /></p> | ||
| + | ==RS+mCherry characterization== | ||
| + | ===Experimental setup=== | ||
| + | <p>In order to provide quality information about the riboswitch function, we fused the MCS+tac promoter+RS construct from the pUC19 to the mCherry protein. The resulting construct was used in a plate reader assay to measure the level of fluorescence as a function of theophylline concentration. After consulting with Dennis Mishler from the Gallivan lab, we learned that full induction of translation should occur at 2mM of theophylline. Thus, we planned a dose response experiment with concentration ranging from 0-8mM theophylline. Several experiments were done, as described in the results section of this page.<br /> | ||
| + | During the second set of experiments, a negative and a positive control were introduced. The negative control is pSB1C3 with an MCS insert. The positive control is a plasmid in which the RS was deleted and replaced with a spacer region with the RBS from the riboswitch.<br /> | ||
| + | All plate reader experiments were done in <em>E. coli </em>TOP10 strain. Briefly, starters were grown over night, diluted 1:100, grown to ~O.D 0.6 and divided into 48 well plates. IPTG was added (to 1mM concentration) to induce the tac promoter and different concentrations of theophylline were added. Controls without IPTG and without theophylline were prepared as well. The samples were incubated at 37 degrees for ~3-4.5 hours before measuring fluorescence in the plate reader.<br /> | ||
| + | ===The positive control=== | ||
| + | The positive control was constructed by doing a PCR reaction to amplify a plasmid with MCS+tac+RS+mCherry without the RS part. The primers were designed with tails which created a spacer region and a PacI restriction site upstream to the RBS from the RS. The PCR products were digested with PacI and self ligated to produce the positive control plasmids. This process was done on several constructs in parallel in order to save time. Some of the constructs contained mutations in the spacer region which should be affecting the RBS strength. Thus, we disregard those differences in the spacer region when looking at our results.<br /> | ||
| + | The spacer region was planned using [http://www.nupack.org/ NUPACK]to create minimal secondary structures around the RBS. Moreover, NUPACK was used in order to compare secondary structures of the RS in fusion to different proteins. Additional information is available on the modeling page.</p> | ||
| + | ==Additional cloning== | ||
| + | <p align="left">The generated RS+mCherry was planned to be used in YES gate #3 of our system. Therefore, it has been cloned downstream to the PLux promoter and submitted as [http://partsregistry.org/wiki/index.php?title=Part:BBa_K784013 BBa_K784013]. Moreover, a positive control has been generated using the method described above and submitted as [http://partsregistry.org/wiki/index.php?title=Part:BBa_K784014 BBa_K784014]. These BioBricks were planned to be assembled upstream to [http://partsregistry.org/Part:BBa_I0462 BBa_I0462] for characterization of induction by both [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] and theophylline.</p> | ||
Revision as of 19:43, 25 September 2012
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Contents |
What is a Riboswitch?
Riboswitches are regulatory elements and can control translation of genes. They consist of a short mRNA sequence that can fold to create a structure. This structure can be changed in the presence of a specific ligand. The regulatory function is achieved if the mRNA structural change can determine if a gene will be translated. In bacteria this can be done by placing the riboswitch before a RBS and ensuring that it will block the ribosome from the RBS, and that after the ligand is added the structural change will allow the ribosome access to the RBS. It is important to note that the function of the RS can be affected by the coding region downstream to it {1,2}.
Fusion PCR
The technique is also known as [http://openwetware.org/wiki/Assembly_pcr assembly PCR]. This technique allows fusing together any two DNA pieces in a precise way, and without having to rely on restriction sites. This is accomplished by careful planning of primes so that for each DNA piece there are 2 primers: in the fused area the primer has a complementary area for the DNA piece and also a complementary area for the second DNA piece; normal primer for the non-fused end {3}.
The first step is to elongate each of the DNA pieces so that each will contain a complementary region for the other DNA piece. This is done by normal PCR {3}.
The second step is to elongate both DNA pieces in the same PCR reaction using only the outer primers. This way the overlapping region of the DNA pieces will act as a third primer, resulting in fused product that contains both DNA pieces {3}.
General design
We used a synthetic theophylline riboswitch, clone 12.1, that was created by S.A Lynch and J.P Gallivan{1}.
We chose to use this kind of regulatory element for the following reasons:
- In our project we are using several different regulatory elements and we need them to work together with little or none crosstalk. Most of the regulatory elements are inducible promoters. We had a hard time finding more inducible promoters that are well defined and don’t crosstalk with the rest of our promoters. So we looked for another kind of regulatory element.
- Riboswitches were discovered only in 2002, and the synthetic ones were created not long after.
- Riboswitches can be useful to future iGEM teams and the rest of the synthetic biology community as an additional and unique regulatory element.
In our project we use Riboswitches with a reporter gene (mCherry) and RNA polymerases: T7, T7*, SP6, T7*(T3),T7*( K1F), T7*(N4) (later notated as T3, K1F and N4){4}.
In order to combine the riboswitch with the different genes we use fusion PCR. We use it because in order for the riboswitch to work the start codon of the gene should be adjacent and downstream to the RBS. This is due to the start codon part in the specific riboswitch structure.
The cassettes that are used in the plasmids are shown in Figure 2.
The Riboswitch is very short (74 bp including the RBS) and it could be a problem with PCR, so we’ve extracted it with 430 bp upstream (part of the pUC19 plasmid) and the long Riboswitch is then 504 bp long. Moreover, the added part from pUC19 contains an MCS (which contains an EcoRI site) and a tac promoter. By adding a PstI site to the end of the RNAP polymerases or mCherry (by carefully planning the primer) we could clone the fusion product into pSB1C3.
The summary of our fusion PCR strategy is presented in Figure 3.
RS+mCherry characterization
Experimental setup
In order to provide quality information about the riboswitch function, we fused the MCS+tac promoter+RS construct from the pUC19 to the mCherry protein. The resulting construct was used in a plate reader assay to measure the level of fluorescence as a function of theophylline concentration. After consulting with Dennis Mishler from the Gallivan lab, we learned that full induction of translation should occur at 2mM of theophylline. Thus, we planned a dose response experiment with concentration ranging from 0-8mM theophylline. Several experiments were done, as described in the results section of this page.
During the second set of experiments, a negative and a positive control were introduced. The negative control is pSB1C3 with an MCS insert. The positive control is a plasmid in which the RS was deleted and replaced with a spacer region with the RBS from the riboswitch.
All plate reader experiments were done in E. coli TOP10 strain. Briefly, starters were grown over night, diluted 1:100, grown to ~O.D 0.6 and divided into 48 well plates. IPTG was added (to 1mM concentration) to induce the tac promoter and different concentrations of theophylline were added. Controls without IPTG and without theophylline were prepared as well. The samples were incubated at 37 degrees for ~3-4.5 hours before measuring fluorescence in the plate reader.
The positive control
The positive control was constructed by doing a PCR reaction to amplify a plasmid with MCS+tac+RS+mCherry without the RS part. The primers were designed with tails which created a spacer region and a PacI restriction site upstream to the RBS from the RS. The PCR products were digested with PacI and self ligated to produce the positive control plasmids. This process was done on several constructs in parallel in order to save time. Some of the constructs contained mutations in the spacer region which should be affecting the RBS strength. Thus, we disregard those differences in the spacer region when looking at our results.
Additional cloning
The generated RS+mCherry was planned to be used in YES gate #3 of our system. Therefore, it has been cloned downstream to the PLux promoter and submitted as [http://partsregistry.org/wiki/index.php?title=Part:BBa_K784013 BBa_K784013]. Moreover, a positive control has been generated using the method described above and submitted as [http://partsregistry.org/wiki/index.php?title=Part:BBa_K784014 BBa_K784014]. These BioBricks were planned to be assembled upstream to [http://partsregistry.org/Part:BBa_I0462 BBa_I0462] for characterization of induction by both [http://partsregistry.org/3OC6HSL 3OC6HSL] and theophylline.
