Part:BBa_K2974700

Strong Promoter (BBa_J23100) Toehold GFP

BBa_J23100 Toehold GFP is a construct designed to be utilized as a color biosensor and to be used in conjunction with part BBa_K2550001. In the 2018 project, Lambert iGEM developed part BBa_K2550000 which was comprised of promoter J23100, a standard Toehold Switch, and a LacZ reporter. However, this part experienced overexpression of the LacZ due to the strong promoter, J23100, of the Anderson series. In order to avoid this problem in Lambert iGEM’s 2019 project, the team looked to replace the J23100 with the medium-strong promoter of the same series, J23106. The team assembled this promoter with a toehold switch and the Green Fluorescent Protein (eGFP) and has been used to compare promoter strength with part BBa_K2974500. When transformed this construct with the complementary trigger sequence, part BBa_K2550000, the colonies produced green fluorescence which was then characterized based on light intensity (lux) values.

Description Toehold switches are synthetic RNA strands that mimic messenger RNA sequences. They contain a complementary recognition sequence (toehold) for a specific sequence of RNA stimuli (trigger), and a ribosomal binding site (RBS) where a ribosome binds to initiate the translation of a reporter protein. The switch has a hairpin loop structure that is formed through binding to complementary sections of its own sequence. When in the presence of the complementary trigger sequence the hairpin loop opens to allow downstream expression. The Ribosomal Binding Site and starting sequence are concealed in the toehold switch until initiated. Switches can provide rapid, convenient, in-field detection that can be developed in both cellular systems and cell-free tests.

In Lambert iGEM’s 2018 project, a T7 Toehold switch was developed to respond to a specific trigger sequence in pathogenic Cholera bacteria, specifically the non-pathogenic gene, ctxB. This cholera-detection system consisted of a strong T7 promoter (BBa_J23100) assembled by the Styczynski Lab at the Georgia Institute of Technology with a De-Novo toehold ribosomal regulator (BBaK2550100) originally designed by the Collins Lab at the Massachusetts Institute of Technology, and the LacZ lactose operon encoding the Beta-galactosidase protein (BBa_I732005). 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 bacterial colonies with the plasmid will appear blue.

In order to induce a strong expression of the downstream reporter, the 2018 team used a strong T7 promoter (BBa_J23100). However, the construct experienced overexpression and induced downstream transcription without the presence of the trigger sequence, leading to false-positive results. In scientific literature, this overexpression is commonly referred to as “leakiness”. To resolve this issue, Lambert iGEM 2019 removed the strong T7 promoter and replaced it with a weaker promoter (BBa_J23106) from the same series of Anderson Promoters.

Additionally, the LacZ operon was replaced with the fluoroprotein enhanced Green Fluorescent Protein (eGFP) to replace the color-based quantification system of detection with a fluorescence-based detection system. In an effort to implement eGFP color expression as a biosensor, Lambert iGEM obtained a De-Novo Toehold Switch construct originally designed in the Collins Lab and assembled with a T7 promoter from the Styczynski Lab at the Georgia Institute of Technology. Promoter BBa_J23106 from the Anderson series was assembled with the De-Novo Toehold Switch from the Collins Lab and the eGFP construct (BBa_E0040) to be applied as a biosensor. When transformed with the distinct compatible RNA sequence, the produced green fluorescent expression can be characterized based on light intensity (lux) values.

Results blah

Sequence and Features

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 728