Difference between revisions of "Part:BBa K2550001"

Latest revision as of 02:24, 18 October 2018

T7 promoter with a trigger sequence to match the toehold sequence of part BBa_K2550000

T7 Toehold LacZ Trigger is an RNA sequence to be used in conjunction with part BBa_K22550000 to induce LacZ expression for a biosensor system. When this trigger RNA sequence is present, it binds to the complementary sequence in the toehold switch and unravels the hairpin loop allowing the reporter protein (LacZ gene) to be expressed; this mechanism produces a blue pigment when inoculated in Xgal. The RNA sequence was obtained from the second entry in the 144 first generation orthogonal toehold switches collection from the 2017 Green etal paper. .

Description

Toehold switches are riboregulators that activate translation in response to a distinct RNA sequence. It is comprised of a switch and a trigger. The switch is composed of a hairpin loop structure that represses translation through its complementary bases in between the ribosome binding site and start codon, which is followed by a 21 nucleotide linker sequence. These sequences ensure that the toehold switch structure will be maintained while coding for low-molecular weight amino acids that would not interfere with the switch’s function. The toehold domains at the beginning of the hairpin are 12 to 18 nucleotides long and are designed to be complementary to the trigger in order to initiate linear RNA binding. The trigger contains complementary sequences to the toehold domain that once it is in the presence of the switch, it will bind to the hairpin stem and unbind the loop. This exposes the ribosome binding site and start codon, allowing translation of the reporter protein to occur.

The lac operon is induced by lactose and IPTG (isopropyl β-D-1-thiogalactopyranoside); this region of the genome is responsible for transporting and metabolizing lactose. Within the lac operon, the gene, LacZ, codes for the B-galactosidase protein. When this protein is expressed, it breaks down Xgal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) into galactose and an insoluble blue pigment. Therefore, when the gene is synthesized in the chassis, the colonies will appear blue. This mechanism was implemented in the toehold construct to identify the presence of specific substances.

Results

Figure 1: (Left) Dual Plasmid Transformation of pSB6A1 T7 Trigger Sequence and pSB6A1 T7 Toehold LacZ on chloramphenicol, carbenicillin, and Xgal antibiotic resistance. (Right) The pSB6A1 T7 Toehold LacZ switch transformed on chloramphenicol, carbenicillin, and Xgal; the construct is not expressing blue color because the trigger is not present.

As seen on the image on the right, the T7 LacZ toehold switch produced no color in the presence of Xgal validating that without the trigger the toehold switch is not induced producing no blue expression from LacZ. However, the dual plasmid transformation demonstrates the role of the trigger when in the presence of the toehold switch. The blue colonies demonstrate that the trigger successfully binded to the toehold domain of the T7 toehold switch allowing the hairpin loop to unbinding producing blue expression from LacZ. Therefore, the trigger sequence was used in a dual plasmid transformation with BBa_K2550000, and as a result, the colonies produced a blue pigment as expected. This proves that the trigger part induces the toehold switch successfully validating its function. Sequence and Features BBa_K2550001 SequenceAndFeatures

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-T7 Toehold LacZ Trigger is an RNA sequence to be used in conjunction with part BBa_K22550000 to induce LacZ expression for a biosensor system. When this trigger RNA sequence is present, it binds to the complementary sequence in the toehold switch and unravels the hairpin loop allowing the reporter protein (LacZ gene) to be expressed; this mechanism produces a blue pigment when inoculated in Xgal. The RNA sequence was obtained from the Georgia Institue of Technology that was number 2 of the 144 generation orthogonal toehold switch collection from the Collins Paper titled, <i>Toehold Switches: De-Novo-Designed Regulators of Gene Expression</i>.
+T7 Toehold LacZ Trigger is an RNA sequence to be used in conjunction with part BBa_K22550000 to induce LacZ expression for a biosensor system. When this trigger RNA sequence is present, it binds to the complementary sequence in the toehold switch and unravels the hairpin loop allowing the reporter protein (LacZ gene) to be expressed; this mechanism produces a blue pigment when inoculated in Xgal. The RNA sequence was obtained from the second entry in the 144 first generation orthogonal toehold switches collection from the 2017 Green etal paper. </i>.
 <br><br>
-<b>Discussion</b>
+<b>Description</b>
 <br><br>
-	Toehold Switches are biosensors that can activate gene expression in response to a chosen RNA sequence, comprised of a switch and a trigger. The switch includes a hairpin loop structure designed to be complementary to the trigger sequence along with a reporter protein downstream. The RBS and starting sequence are concealed in the toehold switch, preventing the reporter from being expressed.
+	Toehold switches are riboregulators that activate translation in response to a distinct RNA sequence. It is comprised of a switch and a trigger. The switch is composed of a hairpin loop structure that represses translation through its complementary bases in between the ribosome binding site and start codon, which is followed by a 21 nucleotide linker sequence. These sequences ensure that the toehold switch structure will be maintained while coding for low-molecular weight amino acids that would not interfere with the switch’s function. The toehold domains at the beginning of the hairpin are 12 to 18 nucleotides long and are designed to be complementary to the trigger in order to initiate linear RNA binding. The trigger contains complementary sequences to the toehold domain that once it is in the presence of the switch, it will bind to the hairpin stem and unbind the loop. This exposes the ribosome binding site and start codon, allowing translation of the reporter protein to occur.
 <br><br>
-The lac operon is induced by lactose and IPTG (isopropyl β-D-1-thiogalactopyranoside); this region of the genome is responsible for transporting and metabolizing lactose. Within the lac operon, the gene, LacZ, codes for the B-galactosidase protein. When this protein is expressed, it breaks down X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) into galactose and an insoluble blue pigment. Therefore, when the gene is synthesized in the chassis, the colonies will appear blue. This mechanism was implemented in the toehold construct to identify the presence of specific substances.
+The lac operon is induced by lactose and IPTG (isopropyl β-D-1-thiogalactopyranoside); this region of the genome is responsible for transporting and metabolizing lactose. Within the lac operon, the gene, LacZ, codes for the B-galactosidase protein. When this protein is expressed, it breaks down Xgal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) into galactose and an insoluble blue pigment. Therefore, when the gene is synthesized in the chassis, the colonies will appear blue. This mechanism was implemented in the toehold construct to identify the presence of specific substances.
 <br><br>
 <b>Results</b>
 <br>
-<img src="https://static.igem.org/mediawiki/parts/7/7e/T--Lambert_GA--JSWORKING.jpeg" style="height:500px;">
+<div style="text-align:center;">
+<img src="https://static.igem.org/mediawiki/parts/a/a4/T--Lambert_GA--JSPICS.png" style="width:500px;">
+</div>
 <br>
-<i> Figure 1: (Left) The biobricked trigger transformation on chloramphenicol antibiotic resistance. (Right Top) The T7 Toehold LacZ switch transformed on chloramphenicol, carbenicillin, and Xgal; the construct is also not expressing blue color because the trigger is not present. (Right Bottom) The blue colonies demonstrated that the tigger induces LacZ expression by binding to the complementary toehold sequence. </i>
+<i> Figure 1: (Left) Dual Plasmid Transformation of pSB6A1 T7 Trigger Sequence and pSB6A1 T7 Toehold LacZ on chloramphenicol, carbenicillin, and Xgal antibiotic resistance. (Right) The pSB6A1 T7 Toehold LacZ switch transformed on chloramphenicol, carbenicillin, and Xgal; the construct is not expressing blue color because the trigger is not present. </i>
 <br><br>
-As seen in the first image, no color is being produced because this transformation singularly included the trigger sequence and the toehold is not present. When the trigger is present, as seen in the top image, the toehold can unbind and the reporter protein is expressed. The trigger sequence was used in a dual plasmid transformation with BBa_K2550000, and as a result, the colonies produced a blue pigment as expected. This proves that the trigger part induces the toehold switch successfully.
+As seen on the image on the right, the T7 LacZ toehold switch produced no color in the presence of Xgal validating that without the trigger the toehold switch is not induced producing no blue expression from LacZ. However, the dual plasmid transformation demonstrates the role of the trigger when in the presence of the toehold switch. The blue colonies demonstrate that the trigger successfully binded to the toehold domain of the T7 toehold switch allowing the hairpin loop to unbinding producing blue expression from LacZ. Therefore, the trigger sequence was used in a dual plasmid transformation with BBa_K2550000, and as a result, the colonies produced a blue pigment as expected. This proves that the trigger part induces the toehold switch successfully validating its function.