Part:BBa_K2916068

Toehold switch for Grapevine Endogenous control 1.1 with superfolding GFP

tbd

Contribution by Evry_Paris-Saclay 2020

BBa_K2916068 is an sfGFP (BBa_I746916) expression cassette under the control of the Grapevine Toehold Switch 1.1 (BBa_K2916050) for sequence-based detection of Vitis vinifera.

As depicted in Figure 1, a toehold switch blocks translation initiation because the RBS and the ATG start codon are embedded in the middle of a hairpin structure maintained by base pairs before and after the start codon [1].

Figure 1. Toehold switches principle.

The hairpin can be unfolded upon binding of a trigger RNA thereby exposing the RBS and the ATG start codon and thus permitting translation of the reporter protein. For this to happen, two important design precautions have to be taken: (i) the start codon should be in frame with the reporter gene, and (ii) there should be no in-frame stop codon before the reporter gene.

This part is a composite part formed by BBa_K2916050, BBa_I746916, BBa_B0010 and BBa_B0012 and was specified with an RFC[10] scar between the parts. The RFC[10] scar TACTAG between the BBa_K2916050 (T7 promoter + grapevine switch 1.1) and BBa_I746916 (sfGFP) introduces a stop codon and leads to premature termination of translation (and thus no expected sfGFP production).

Moreover, the role of the trigger RNA is to bind to the toehold switch and thus unfold the hairpin structure thereby exposing the RBS and the ATG start codon and thus permitting translation of the reporter protein. The binding between the trigger and the switch is dictated by RNA-RNA interactions between complementary base pairs.

In BBa_K2916050 (which is the T7 promoter together with grapevine switch 1.1 used to build the composite part BBa_K2916068), the trigger binding region is in the same direction as in the grapevine genome and of the trigger sequences derived from (BBa_K3453081 and BBa_K3453083). As a result, this trigger / switch pair does not interact and is not functional. However, the hairpin structure of the grapevine switch 1.1 is predicted to be unfolded by the reverse complements of the two triggers BBa_K3453082 and BBa_K3453084 (Table 1).

Table 1. Grapevine triggers.
Label	Part number	Transcriptional units’ part numbers	Source
Grapevine trigger 'short'	BBa_K3453081	BBa_K3453181	EPFL 2019 iGEM team personal communication
Grapevine trigger 'short reverse'	BBa_K3453082	BBa_K3453182	Reverse complement of BBa_K3453081
Grapevine trigger 'long'	BBa_K3453083	BBa_K3453183	designed by the EPFL 2019 iGEM team and its sequence was deduced from “Amplification” page of the EPFL 2019 wiki where is described how the triggers were designed: the grapevine trigger was PCR amplified using as template the Vitis vinifera TrnL-UAA and the forward primers EC_fwd1 or EC_fwd1_T7 and the EC_rev1 reverse primer (it should be noted that the sequences of all reverse primers listed in the additional materials of this page are given as 3’-5’ sequences and not in the conventional 5’-3’ orientation).
Grapevine trigger 'long reverse'	BBa_K3453084	BBa_K3453184	Reverse complement of BBa_K3453083

To confirm the above observations, BBa_K2916068 was synthesized and assembled in the low copy plasmid pSB3T5. The trigger sequences (Table 1) were placed under the control of the T7 promoter and followed by the strong SBa_000587 synthetic terminator for the T7 RNA polymerase [3] and the resulting transcriptional units were synthesized and assembled in the high copy plasmid pSB1C3.

For toehold switch characterisation we decided to use E. coli BL21 Star™(DE3) cells (Thermo Fisher Scientific), genotype F- ompT hsdSB (rB-mB-) gal dcm rne131 (DE3). As all BL21(DE3) E. coli strains, these cells contain the T7 RNA polymerase under control of the lacUV5 promoter and thus require IPTG to induce expression. The particularity of BL21 Star™(DE3) cells is that they contain a truncated version of the RNaseE gene (rne131) that leads to reduced level of mRNA degradation and thus increased RNA stability. For fluorescence measurements, E. coli cells containing switch and trigger plasmids were first grown overnight in 96-deep-well plates containing 1 mL of LB medium supplemented with 5 µg/mL tetracycline and 17.5 µg/mL chloramphenicol, then diluted by 40x into similar media. Upon reaching early log-phase, cells were further diluted 20x in 100 µL of LB medium supplemented with 5 µg/mL tetracycline, 17.5 µg/mL chloramphenicol and 10 µM IPTG in an opaque wall 96-well polystyrene microplate, the COSTAR 96 (Corning). The plate was incubated at 37°C at 200 rpm and the sfGFP fluorescence (λexcitation 483 nm and λemission 530 nm) and OD600nm were measured every 10 min for 24 hours in a CLARIOstar (BMGLabtech) plate reader. Fluorescence values were normalised by OD600nm and, using the calibration curves presented on the ‘Measurement’ page of our wiki, we converted the arbitrary units into Molecules of Equivalent FLuorescein (MEFL) / particle.

The results presented in figures 2, 3 and 4 show that this part is not functional, as expected from the above observations: we were also unable to detect any significant sfGFP expression from the BBa_K2916068. This confirms that the stop codon introduced by the RFC[10] scar TACTAG plays an important negative role in the function of the toehold switch, as the translation initiated at the AUG codon embedded in the hairpin structure is prematurely stopped before sfGFP translation.

The results presented in Figure 2 show 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 harbouring the grapevine toehold switch 1.1 in BBa_K2916068 and the 'short' and 'long' grapevine triggers in both forward and reverse orientation (BBa_K3453181, BBa_K3453182, BBa_K3453183 and BBa_K3453184). The negative controls have been performed with an empty pSB3T5, pSB1C3 (no trigger) and BBa_K3453103 (no promoter, no RBS) and the positive controls with BBa_K3453104 and BBa_K3453105. 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 grapevine toehold switch 1.1 in BBa_K2916068 in the presence of the 'short' and 'long' grapevine triggers in both forward and reverse orientation (BBa_K3453181, BBa_K3453182, BBa_K3453183 and BBa_K3453184) compared to the MEFL / Particle value in the absence of the trigger.

Figure 4: Pictures of E. coli BL21 Star™(DE3) cells harbouring this part and in the presence of the 'short' and 'long' grapevine triggers in both forward and reverse orientation (BBa_K3453181, BBa_K3453182, BBa_K3453183 and BBa_K3453184). The negative control was performed with an empty pSB1C3 (no trigger).

To circumvent the problems described above, we constructed BBa_K3453170 in which the RFC[10] scar TACTAG was removed by site directed mutagenesis on BBa_K2916068.

In addition, as the situation turned out to be more complex than expected, we designed two derivatives of the grapevine toehold switch 1.1 denoted 1.1.2 and 1.1.3, three truncated versions laking the trigger binging sites, as well as their cognate sfGFP or sfGFP-LVAtag expression cassettes.

These parts are listed in Table 2 and the results of their characterisation summarised in Figure 5. All details are available on their individual pages in the registry and the full story can be read on the 'Contribution' page of our wiki.

Table 1. Grapevine toehold switches 1.1 and derivatives.
Label	Toehold Switch Part Number	Toehold Switch + sfGFP Part Number	Toehold Switch + sfGFP-LVAtag Part Number
Grapevine VvTrnL-UAA Toehold Switch 1.1	BBa_K2916050	BBa_K2916068 & BBa_K3453170	BBa_K3453171
Truncated Grapevine VvTrnL-UAA Switch 1.1 (no trigger binding site)	BBa_K3453072		BBa_K3453172
Grapevine VvTrnL-UAA Toehold Switch 1.1.2	BBa_K3453073	BBa_K3453173	BBa_K3453174
Truncated Grapevine VvTrnL-UAA Switch 1.1.2 (no trigger binding site)	BBa_K3453075		BBa_K3453175
Grapevine VvTrnL-UAA Toehold Switch 1.1.3	BBa_K3453076	BBa_K3453176	BBa_K3453177
Truncated Grapevine VvTrnL-UAA Switch 1.1.3 (no trigger binding site)	BBa_K3453078		BBa_K3453178

Figure 5. In vivo characterization of sfGFP expression by E. coli BL21 Star™(DE3) cells harbouring the grapevine toehold switches 1.1 (Table 2) and the 'short' and 'long' grapevine triggers in both forward and reverse orientation (BBa_K3453181, BBa_K3453182, BBa_K3453183 and BBa_K3453184). The negative controls have been performed with an empty pSB3T5, pSB1C3 (no trigger) and BBa_K3453103 (no promoter, no RBS) and the positive controls with BBa_K3453104 and BBa_K3453105. The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.

BBa_K3453170, BBa_K3453171, BBa_K3453173, BBa_K3453174, BBa_K3453176 and BBa_K3453177 are technical improvements of BBa_K2916068. However, all these six improved parts are functional in the presence of a trigger artificially expressed in E. coli in reverse orientation. They are not able to detect the presence of an RNA naturally expressed in grapevine because the trigger binding site is not in a compatible orientation. But that is another story, and we invite the reader to discover it on the 'Improvement' page of our wiki.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 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 53
  Illegal SapI.rc site found at 144