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− | To detect influenza A, Polymerase Basic Protein 2 (PB2) gene is used as a positive control as it is influenza A-specific. Further subtyping requires a subtype-specific RNA that can also fulfil the criteria for being a good toehold switch. We downloaded the latest influenza gene sequences from the Influenza Research Database and inputted to our program to generate switches to detect H5, H7, N1, N9 and PB2 RNAs. The sequences used are listed below: | + | To detect influenza A, Polymerase Basic Protein 2 (PB2) gene is used as a positive control as it is influenza A-specific. Further subtyping requires a subtype-specific RNA that can also fulfil the criteria for being a good toehold switch. We downloaded the latest influenza gene sequences from the Influenza Research Database and inputted to our program to generate switches to detect H5, H7, N1, N9 and PB2 RNAs. The sequences used are listed below (Type of flu/ region of origin/ number of lineage/ year of isolation): |
<center><img src="https://static.igem.org/mediawiki/2017/e/e7/Experimap.jpg" style="width:540px;height:360px;"></center> | <center><img src="https://static.igem.org/mediawiki/2017/e/e7/Experimap.jpg" style="width:540px;height:360px;"></center> | ||
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3 toehold switches with “good” predicted performance were chosen to target each RNA (For more information, please visit <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model">RNA thermodynamics modelling page</a>). For example, the three switches are named as H5-1, H5-2 and H5-3 for H5 RNA detection. The figure above shows the detection region of each toehold switch. Before constructing the toehold switches, we ensured all the switches passed our modelling criteria. | 3 toehold switches with “good” predicted performance were chosen to target each RNA (For more information, please visit <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model">RNA thermodynamics modelling page</a>). For example, the three switches are named as H5-1, H5-2 and H5-3 for H5 RNA detection. The figure above shows the detection region of each toehold switch. Before constructing the toehold switches, we ensured all the switches passed our modelling criteria. | ||
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<p><h3>Construction of toehold switch and trigger-expressing plasmid</h3></p> | <p><h3>Construction of toehold switch and trigger-expressing plasmid</h3></p> | ||
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | The upper picture showed the general structure of our toehold switch. mRFP was chosen as the reporter of our toehold switches because it is very distinguishable by naked eyes while at the same time it can be quantified by measuring the fluorescence signal using a plate reader. After having the toehold switch sequences generated by our program, we linked it with the reporter sequence and synthesized them using IDT’s sponsored gBlock synthesis service. The gBlocks were used as template and amplified by PCR. The bands with correct size were gel-purified. We inserted the purified PCR products into pSB4C5 (for switch) or pSB1K3 (for trigger) using restriction cut and ligation. Sequencing results confirmed all 30 constructs were cloned. | + | <center><img src="https://static.igem.org/mediawiki/2017/d/dc/CUHK_toeholdstructure.jpg" width="50%" height="auto"></center> |
+ | The upper picture showed the general structure of our toehold switch (For detailed structure, please visit our <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model"> modelling page</a>). mRFP was chosen as the reporter of our toehold switches because it is very distinguishable by naked eyes while at the same time it can be quantified by measuring the fluorescence signal using a plate reader. After having the toehold switch sequences generated by our program, we linked it with the reporter sequence and synthesized them using IDT’s sponsored gBlock synthesis service. The gBlocks were used as template and amplified by PCR. The bands with correct size were gel-purified. We inserted the purified PCR products into pSB4C5 (for switch) or pSB1K3 (for trigger) using restriction cut and ligation. Sequencing results confirmed all 30 constructs were cloned. | ||
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<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | These two backbones with different Ori and antibiotic resistance genes were used because they will be used in the following experiments. Two co-transformed plasmids should not have the same type of origin of replication (Ori), or otherwise, they will compete for the replication machinery and affect the copy number. In addition, having two different antibiotic resistance genes avoid dropping out of either one of the plasmids during selection. | + | These two backbones with different Ori and antibiotic resistance genes were used because they will be used in the following experiments. Two co-transformed plasmids should not have the same type of origin of replication (Ori), or otherwise, they will compete for the replication machinery and affect the copy number. A higher copy number is chosen for the trigger plasmid to ensure trigger expression is in excess in cells. In addition, having two different antibiotic resistance genes avoid dropping out of either one of the plasmids during selection. |
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Revision as of 06:45, 28 October 2017