Part:BBa_K2550000:Design

T7 promoter Toehold Ribosome Switch with LacZ expression

T7 Toehold LacZ is a construct that was developed to be applied as a biosensor. The part BBa_I732005 was submitted by 2007 UTSC iGEM that singularly included the LacZ lactose operon encoding the Beta-galactosidase protein. In order to utilize LacZ as a biosensor mechanism, Lambert iGEM obtained a LacZ toehold construct assembled with a T7 promoter from the Styczynski Lab at Georgia Institute of Technology. When assembled with a distinct RNA sequence that is complementary to the trigger sequence, the color can be characterized with the produced blue pigment expression. Toehold Switches are biosensors that can activate gene expression in response to a chosen RNA sequence. It is comprised of a switch and a trigger. The switch is comprised of a hairpin loop that is 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. When the trigger RNA sequence is present, it will bind to the complementary sequence in the toehold switch and unbind the hairpin loop allowing the reporter protein to be expressed. T7, part BBa_I719005, is a strong constitutive promoter that expresses proteins in the presence of T7 polymerase. This promoter was derived from the T7 bacteriophage which commonly contaminates Escherichia coli. 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. The Toehold sequence was obtained from the 144 first generation orthogonal toehold switches collection from the 2017 Collins paper titled “...” 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 can be used as a biosensor or reporter to identify the presence of specific substances. 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 Georgia Institute of Technology and was used to eliminate the illegal EcoRI site in the LacZ operon.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Needs to be used in low to medium copy plasmids only

References

Alexander A. Green, Pamela A. Silver, James J. Collins, Peng Yin, Toehold Switches: De-Novo-Designed Regulators of Gene Expression, Cell, Volume 159, Issue 4, 2014, Pages 925-939, ISSN 0092-8674, https://doi.org/10.1016/j.cell.2014.10.002