Part:BBa_K2974400

T7 C. elegans Trigger

The T7 Toehold Trigger is an RNA sequence to be used in conjunction with part BBa_K2974316 to induce expression of GFP within a biosensor system. When this trigger RNA sequence is present, it binds to the complementary sequence in the toehold switch and unravels the hairpin loop allowing the reporter protein (GFP) to be expressed; As a result, this mechanism produces fluorescence. The complementary sequence was calculated using NUPACK (Nucleic Acid Package) software, and the trigger was experimentally tested with part BBa_K2974316 to determine whether the toehold’s secondary structure formed correctly and whether the RBS was sufficiently enclosed in the upper loop.

Description

Toehold switches are riboregulators that activate translation in response to a distinct RNA sequence. It is comprised of a switch and a trigger. The switch is composed of a hairpin loop structure that represses translation through its complementary bases in between the ribosome binding site and start codon, which is followed by a 21 nucleotide linker sequence. These sequences ensure that the toehold switch structure will be maintained while coding for low-molecular weight amino acids that would not interfere with the switch’s function. The toehold domains at the beginning of the hairpin are 12 to 18 nucleotides long and are designed to be complementary to the trigger in order to initiate linear RNA binding. The trigger contains complementary sequences to the toehold domain that once it is in the presence of the switch, it will bind to the hairpin stem and unbind the loop. This exposes the ribosome binding site and start codon, allowing translation of the reporter protein to occur.

Results
The T7 C. Elegans Trigger was assembled into pSB1A3, a high copy plasmid. After obtaining the C. elegans Trigger gBlock, we digested the trigger insert using EcoRI-HF and PstI-HF. We confirmed that the insert digest worked using gel electrophoresis. After ligating the insert into a pSB1A3 vector digest and transforming with DH5α E. coli competent cells, we purified the DNA. The C. elegans trigger DNA was then sent to sequencing, which came back successful. Sequencing results (see experience page) along with an additional gel confirmed the successful assembly of our Trigger insert.

Figure 1: The white trigger colonies can be seen on this plate, showing that the trigger successfully ligated into pSB1A3. There is some RFP contamination present, but this was not a deterrant since we only chose colonies expressing GFP for the continuation of our workflow.

Figure 2: The gel electrophoresis demonstrates the successful Trigger insert digest. In lane 4 and 6, the 2-log ladder and the 100bp ladder respectively are present. The trigger insert band, which is in lane 5, is faintly shown around 160 bp.

Additionally, the functionality of our toehold was confirmed by using the Miniprep products from both the Toehold Switch Assembly and the Trigger Assembly to perform a dual plasmid transformation on carbenicillin/chloramphenicol-resistant LB plates. We decided to use BL21(DE3) E. coli competent cells from NEB because they allow for T7 expression. After growing for 48 hours in the incubator at 37ºC, we observed GFP expression from the transformed dual plasmid colonies.

After obtaining a purified DNA product of the Dual Plasmid, we performed a Restriction Digest in order to demonstrate that both vectors were successfully cloned into BL21 (DE3) E. coli competent cells. In lanes 1, 2, and 5, digests of colonies 1, 2, and 3 all show the separate vectors respectively. The larger band, C. elegans toehold in psB3C5 , is shown around 2800 base pairs, and the smaller band, T7 C. elegans trigger in psB1A3, is shown around 2155 base pairs

Figure 3: Dual Plasmid transformation colonies are shown here. Since the toehold is now in the presence of the trigger, the competent cells expressed a green fluorescence.

Figure 4: Gel showing the digests of the Dual Plasmid Transformation of T7 Trigger Sequence and T7 Toehold GFP. These show the expected results of our constructs.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 159
  Illegal NotI site found at 6
  Illegal NotI site found at 153
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 69