Part:BBa_K4432105
PANTR1 Toehold Switch Sensor 05 with sfGFP-LVAtag
This part is an sfGFP-LVAtag (BBa_K2675006) expression cassette under the control of the PANTR1 Toehold Switch N°5 (BBa_K4432005) for sequence-based detection of PANTR1, a long non coding (lnc) RNA overexpressed in different types of cancer cells.
Usage and Biology
A toehold switch [1] (Figure 1) is an RNA-based device is composed of several parts which are essential for the efficient functioning of our biosensor :
- a Trigger Binding Site (TBS) which is a sequence complementary to the RNA target to be detected
- a Ribosome Binding Site (RBS) which allows the start of the translation only when the toehold switch is unfolded
- a start codon AUG which constitutes the start of the translation
- a linker which constitutes a bridge between the Start codon and our gene of interest
- a repressed gene of interest that is translated into a protein only when the hairpin is unfolded and the RBS exposed
The functioning comes as follows :
The binding between the RNA biomarker and the toehold switch unfolds the hairpin structure. The RBS becomes accessible now so that the translation can take place, and the initially repressed gene of interest can then be translated into a protein.
Figure 1. Principle of the toehold switch (inspired from iGEM Evry Paris-Saclay 2020).
In this composite part sfGFP-LVAtag (BBa_K2675006) was equipped by the synthetic PANTR1 Toehold Switch N°5 (BBa_K4432005) and placed under the control of the T7 promoter (BBa_K2150031) and of the strong SBa_000587 synthetic terminator (BBa_K3453000).
It was assembled by Golden Gate in the low copy plasmid pSB3T5. For this, a fragments containing the BBa_K4432005 was synthesized flanked by BsaI type IIS restriction sites, and then inserted into BBa_K3453101, which contains a Golden Gate adapter with BsaI sites upstream of the sfGFP-LVAtag.
The corresponding trigger sequence of this toehold switch is the PANTR1 Trigger N°5 (BBa_K4432205). It was placed under the control of the T7 promoter and followed by the strong SBa_000587 synthetic terminator for the T7 RNA polymerase and the resulting transcriptional unit was synthesized and assembled in the high copy plasmid pSB1C3.
in vivo experimental characterization
For toehold switch characterization, cells of E. coli BL21 Star™(DE3) (Thermo Fisher Scientific) were used. This strain was chosen, first of all because it contains the T7 RNA polymerase gene, which is under the control of the lacUV5 promoter and requires IPTG to induce expression, and second because the it has the particularity to contain a truncated version of the RNAseE gene (rne131), which reduces the level of RNA degradation and thus increases RNA stability. E. coli cells containing both the toehold switch plasmid and the trigger plasmid were grown overnight at 37 °C at 200 rpm in 96-deep-well plates with 1 mL of LB, in which was added 5 µg/mL tetracycline and 17.5 µg/mL chloramphenicol. The cells were then diluted by 40 times in the same media and after 4 hours incubation at 37°C at 200 rpm, they were further diluted by 20 times in media containing also 10 µM IPTG in an opaque wall 96-well polystryrene microplate (COSTAR 96, Corning). The plate was then incubated at 37°C at 200 rpm. The sfGFP fluorescence and optical density (OD) were measured every 10 minutes for 24 hours, in a CLARIOstar (BMGLabtech) plate reader. We measured fluorescence by measuring λexcitation 488 nm and λemission 530 nm, and OD was measured at 600nm. Fluorescence values were normalized by OD600. Furthermore, the arbitrary units have been converted into Molecules of Equivalent FLuorescein (MEFL) / particle (more details about the calibration curves on the the ‘Measurement’ page of our wiki).
The results presented in figure 2 show that this PANTR1 toehold switch n°5 is functional and behaves as expected. When tested against its cognate trigger, this toehold switch showed fluorescence with a mean comparable to that of the positive controls (Figure 2) suggesting that we leveraged the full capacity of the expression cassette. The ON/OFF ratio of this toehold switch was good (15).
Additional crosstalk experiments were carried out to evaluate its toehold switches behavior in the presence of all available PANTR1 triggers (BBa_K4432201, BBa_K4432202, BBa_K4432203, BBa_K4432204, BBa_K4432205, BBa_K4432206, BBa_K4432207, BBa_K4432407 BBa_K4432607, BBa_K4432208). Results presented in Figures 2 and 3 revealed that Switch n°5 crosstalks with Switch n°6. It showed a significant fluorescence with cognate trigger n°5 and non-cognate trigger n°6. These results are not unexpected as switches n°5 and n°6 have an overlap of 31/35 nucleotides.
The results presented in Figures 2 and 3 show also that the sfGFP expression was readily detected in the positive controls and that the two RBS (BBa_K2675017 and BBa_K3453005) have comparable strength. As expected, no fluorescence was detected in the negative controls as either the sfGFP gene was not present or the promoter and the RBS were absent.
Figure 2. In vivo characterization of sfGFP expression by E. coli BL21 Star™(DE3) cells carrying the PANTR1 toehold switch n°5 and its cognate trigger. The negative controls have been performed with an empty pSB3T5 and BBa_K3453103 (no promoter, no RBS), and pSB1C3 (no trigger) for every Switch. The positive controls have been performed with BBa_K3453104 (custom made RBS) and BBa_K3453105 (standard toehold switch RBS stem-loop). The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.
Figure 3. MEFL/Particle fold changes of the PANTR1 toehold switch n°5 in the presence of each of all the other triggers compared to the MEFL/Particle value in the absence of the trigger.
References
[1] Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell (2014) 159, 925-939.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 150
- 1000COMPATIBLE WITH RFC[1000]
| None |