Team:Lambert GA/Results


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 Switch BBa_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 2: (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 3: 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.

Cholera Toehold Switch

To test the functionality of our Cholera toehold switches, we first performed dual plasmid transformations by electroporating 10 ng/ul of each switch and trigger plasmid into BL21 E. coli. In addition, we performed single transformations on each of the switches to test the on-off functions of our toehold switches. All three of our Cholera switch transformations presented blue colonies. We have hypothesized that this is due to the leakiness of the switches in response to the strength of the T7 promoter causing the blue expression of LacZ when in the presence of Xgal. In addition, the dual plasmid transformations were unsuccessful. This could be due to the low time constants that were produced during electroporation causing the BL21 cells to not have their pores open for enough time preventing efficient uptake of the plasmids. Due to these inconclusive results, Lambert iGEM performed a chemical transformation with these toehold switches.
Figure 4: Unsuccessful Electroporation- Plates on top are the single switch transformations. Plates on the bottom are the dual plasmid transformations.

Figure 5: Chemical Transformation- Plates on the top are single switch transformations of the Cholera switches. The plates on the bottom are the dual plasmid transformations of Cholera 3, 2, and 1.

Unsuccessful: Leakiness

Due to the inconclusive results from the electroporation, Lambert iGEM performed a chemical transformation with our Cholera toehold switches and triggers in BL21 E. coli. For the dual plasmid transformation, 50 ng/ul of both switch and trigger plasmids were used with a total of 100 ng of plasmid. Both Cholera 1 and 2 single switch transformations show a strong blue expression demonstrating the same leakiness presented in the electroporation results. However, the Cholera 3 switch was grown on a chloramphenicol plate in the absence of Xgal causing these cells to not express the blue pigment from LacZ. In addition, unlike the dual plasmid results from the electroporation, the chemically transformed cells grew and expressed blue pigment at a lower efficiency than the single switch transformations.

Sequencing: unsuccessful

The sequencing results for the Cholera switches show that the Cholera 1 and 2 switches contain the toehold switch from the 2017 Collins paper titled “ Toehold Switches: De-Novo-Designed Regulators of Gene Expression”. Therefore, our Cholera switches did not contain the correct sequences.