Part:BBa_K2974316
Medium Promoter (BBa_J23106) C. elegans Toehold GFP
BBa_J23106 C. elegans toehold was built using BBa_K2550202 as the stem-loop domain for the toehold, and the target sequence domain is derived from the C. elegans lin-4 gene pre-mRNA primary transcript F59G1.6 (non-toxic). This part encodes Switch Number 1 of the C. elegans Toehold Series developed by Lambert iGEM. The described structure was optimized in design using the parameters described by Pardee et al. and validation using the toehold designer software developed by To et al. The design of this switch involved consideration of numerous components including free energy of RBS-Linker, MFE of various structures, illegal stop codons, etc. in order to optimize the secondary structure sequence. The corresponding sequence was then processed using the NUPACK (Nucleic Acid Package) to determine whether the appropriate secondary structure formed and whether the RBS was sufficiently enclosed in the upper loop. Following analysis of the sequence and structure, this part was experimentally tested.
Description
Toehold switches are RNA-based riboregulators functioning as switches that can activate/inhibit reporter expression using a synthetic construct. In the presence of a reverse complementary RNA trigger sequence, reporter expression can be activated through the binding of the trigger sequence to the toehold switch, resulting in the uncoupling of the secondary structure, allowing the RBS to be freed from the "closed" position, allowing for translation of the reporter protein. Toehold switches are advantageous as arbitrary sequences can be utilized to regulate expression of a reporter, serving as unique tools for incorporation as biosensors.
Results
The Medium Promoter C. elegans Toehold GFP BioBrick (BBa_K2974316) was assembled into pSB3C5, a medium copy plasmid. After obtaining the C. elegans Toehold GFP gBlock, we digested the Toehold insert using EcoRI-HF and PstI-HF. We confirmed that the insert digest worked using gel electrophoresis. After ligating the insert into a pSB3C5 vector digest and transforming with DH5α E. coli competent cells, we purified the DNA. The Toehold DNA was then sent to sequencing, which came back successful. Sequencing results along with an additional gel confirmed the successful assembly of our Toehold insert.
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 1: 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 2: 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 688
| None |