Part:BBa_K2550000:Experience

UPDATE 2019=

Update 2019 A new part has been designed by Lambert 2019 BBa_K2974500 to improve the function of the toehold switch by eliminating over expression when the trigger sequence is not present. It is hoped that this will lead to less false positives. The sequence has been assembled. Results from characterization experiments had not been completed by the registry freeze. Data will be added after the Jamboree. Part BBa_K2550000 should not be used as it has significant leakiness in over expression. A New Improved part BBa_K2974500 should be used in it's place. Lambert 2019 experience.

Figure 1: Toehold structure implemented in the T7 Toehold LacZ biobrick that was developed in the NUPACK (Nucleic Acid Package) software which confirmed the shape of the toehold sequence.

The toehold sequence, part BBa_K2550100, is 108 bp long and was obtained from the 144 first generation orthogonal toehold switches collection from the 2017 Collins paper titled Toehold Switches: De-Novo-Designed Regulators of Gene Expression. This distinct riboregulator follows the T7 promoter that is assembled in the T7 Toehold LacZ biobrick. This image above is a visual representation of this unique toehold structure.

Figure 2: The T7 Toehold LacZ biobrick transformed on a chloramphenicol and Xgal plate expressing blue pigment without trigger sequence as a result of the strong T7 promoter (Image on the left). T7 Toehold LacZ and trigger sequence transformed in a dual plasmid transformation on a carbenicillin, chloramphenicol, and Xgal plate that is expressing a blue pigment due to presence of trigger sequence (Image on the right).

T7, part BBa_I719005, is a strong constitutive promoter derived from the T7 bacteriophage that expresses proteins in the presence of T7 polymerase. It was observed that the toehold expressed a blue pigment when inoculated into Xgal and Luria Broth. Although a lighter shade than when fully induced, we hypothesize that this pigment is due to toehold leakiness as a result of the strength of the T7 promoter.

Sequencing Analysis

Figure 3: Image showing the sequencing results of the T7 Toehold LacZ construct. Annotated in a white arrow is the biobricked T7 promoter (BBa_I719005); this is followed by the biobricked toehold sequence which was used in the NUPACK software to illustrate the functioning Toehold Switch (BBa_K2550100). Illustrated in the maroon arrow is the biobricked LacZ gene (BBa_I732005) sequence that justified that the one base pair wobble mutation was successful.

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