Team:Lambert GA/Results

CALM ELECTROPEN
BACKGROUND DATA & METHODS RESULTS DISCUSSION COLOR Q SUPPLEMENTARY REFERENCES
COLOR Q CHROME Q REFERENCES
PRINCIPLES & DESIGN MODEL & THEORY PRODUCT DESIGN REFERENCES
INTEGRATED HUMAN PRACTICES EDUCATION & ENGAGEMENT COLLABORATIONS
BASIC PARTS COMPOSITE PARTS
DESCRIPTION BACKGROUND DESIGN EXPERIMENTS LAB NOTEBOOK INTERLAB RESULTS DEMONSTRATE THE VISION
TEAM MEMBERS ATTRIBUTIONS

R E S U L T S



































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.

Lambert iGEM used sequences and DNA shared from the Styczynski Lab at the Georgia Institute of Technology to build a T7, Toehold, LacZ switch to be applied as a biosensor. We tested the Biobricked T7 Toehold LacZ Switch BBa_K2550000, the Biobricked T7 Trigger Sequence BBa_K2550001 T7 Toehold LacZ Switch obtained from GATech, and the pSB6A1 T7 Trigger on Luria broth plates with chloramphenicol, carbenicillin, and Xgal antibiotic resistance.


Figure 1: (Top left) Biobricked T7 Toehold LacZ SwitchBBa_K2550000 transformed on chloramphenicol, carbenicillin, and Xgal. (Top right) Biobricked Trigger Sequence BBa_K2550001 transformed on chloramphenicol antibiotic resistance. (Bottom left) pSB6A1 T7 Toehold LacZ Switch transformed on chloramphenicol, carbenicillin, and Xgal. (Bottom right) Dual plasmid with biobricked toehold sequence and trigger sequence.

The dual plasmid transformation using the pSB6A1 trigger and biobricked toehold was successful. Since the blue expression was only evident on the dual plasmid plate and not on the plate that contained the toehold, the trigger is successful in inducing LacZ gene expression.

The transformations of the biobricked toehold construct were unsuccessful because they produced a blue color. The blue might be a result of the orthogonal switch not binding as expected or a result of overexpression from a too strong promoter. Since the trigger sequence was not transformed with the construct, the colonies should appear white as the toehold only reveals the RBS and starting sequence of LacZ when the trigger sequence. This could be due to a leaky expression of LacZ under the strong T7 promoter. The dual plasmid transformation confirmed that neither the biobricked trigger nor the biobricked toehold sequence worked in conjunction with each other although they worked in conjunction with similar parts not in pSB1C3.


Figure 3: (Right) Dual Plasmid Transformation of T7 Trigger Sequence and T7 Toehold LacZ on chloramphenicol, carbenicillin, and Xgal antibiotic resistance. (Left) T7 Toehold LacZ transformation on chloramphenicol antibiotic resistance. These show the expected results of our constructs.



Figure 4: Sequencing results of the trigger sequence that confirmed the part was correct.

We aligned the sequencing results to the original trigger sequence obtained from the second entry in the 144 first generation orthogonal toehold switches collection from the 2017 Green etal paper. Since the sequences matched after alignment, this confirmed that the trigger sequence was correctly cloned.



Figure 2: 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.