Team:Lambert GA/Results

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BACKGROUND DATA & METHODS RESULTS DISCUSSION COLOR Q SUPPLEMENTARY REFERENCES
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PRINCIPLES & DESIGN MODEL & THEORY PRODUCT DESIGN REFERENCES
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Proof of Concept Results


In order to utilize LacZ color expression as a biosensor mechanism, Lambert iGEM obtained a LacZ toehold construct assembled with a T7 promoter from the Styczynski Lab at the Georgia Institute of Technology. When assembled with a distinct RNA sequence that is complementary to the trigger sequence, the LacZ operon is induced and subsequently breaks down Xgal and produces galactose and a blue pigment. This color can then be characterized by the shade of the blue pigment expression. This part is transformed in pSB3C5 instead of pSB1C3 because when LacZ is induced in a high copy plasmid such as pSB1C3, it drains the metabolism of a cell.



The construct above displays the proof of concept T7 LacZ switch and trigger. The switch consists of T7 promoter, a strong constitutive promoter, along with LacZ as the reporter gene. The trigger was cloned into a high copy plasmid while the switch was cloned into a low copy plasmid to ensure the replication of two different plasmids for the E. coli cell to produce proportional amounts.




The T7 Toehold LacZ biobrick 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 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).


As seen in the figure above, it was observed that the toehold expressed a blue pigment when inoculated into Xgal and Luria Broth (image on the left). Although a lighter shade than when fully induced (image on the right), we hypothesize that this apparent pigment is due to toehold leakiness as a result of the strength of the T7 promoter. The Toehold sequence used in this construct 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”. Following this unique toehold sequence is the LacZ operon. We introduced a base pair wobble in the LacZ gene that substituted an Adenine for a Guanine. The wobble mutation sequence was obtained from the Styczynski Lab at the Georgia Institute of Technology and was used to eliminate the illegal EcoRI site in the LacZ operon.


In order to utilize LacZ color expression as a biosensor mechanism, Lambert iGEM obtained a LacZ toehold construct assembled with a T7 promoter from the Styczynski Lab at the Georgia Institute of Technology. When assembled with a distinct RNA sequence that is complementary to the trigger sequence, the LacZ operon is induced and subsequently breaks down xgal and produces galactose and a blue pigment. This color can then be characterized by the shade of the blue pigment expression.


Figure 1: The T7 Toehold LacZ biobrick 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 in a dual plasmid transformation on a carbenicillin, chloramphenicol, and Xgal plate that is expressing a blue pigment due to the presence of trigger sequence (Middle Image and Right Image).

As seen in the figure above, it was observed that the toehold expressed a blue pigment when inoculated into xgal and Luria Broth (image on the left). Although a lighter shade than when fully induced (image on the right), we hypothesize that this apparent pigment is due to toehold leakiness as a result of the strength of the T7 promoter. The Toehold sequence used in this construct 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”. Following this unique toehold sequence is the LacZ operon. We introduced a base pair wobble in the LacZ gene that substituted an Adenine for a Guanine. The wobble mutation sequence was obtained from the Styczynski Lab at the Georgia Institute of Technology and was used to eliminate the illegal EcoRI site in the LacZ operon.