Results

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Introduction

The original tRNA-Mimicking Sequence (TMS) comes from the work conducted by Paul et al. (2020). Here, they achieve ligand-selective protein translation-inhibition by creating a tRNA-like structure modified by insertion of an aptamer sequence. Across our project, we tried to reproduce part of the work by Paul et al. (2020), and we expanded the TMS family with several new structures to be used as biobricks applicable mainly for biosensor production, and specifically for PFOA detection in water.

Validation of the TMS (GFP)

The first step of our project was the reproduction of the TMS (GFP) from Paul et al. (2020), BBa_K4811002. This was tested as the composite part BBa_K4811018 in E. coli BL21(DE3), where the TMS is induced by addition of Isopropyl β-d-1-thiogalactopyranoside (IPTG). This construct was USER cloned into the high copy number pUC19 backbone for testing (see Design and Engineering). A reporter, consisting of BBa_K4811005 was USER cloned into the low copy number pACYC184 backbone. This has GFP under the control of the Ptet promoter, and therefore is induced by anhydrotetracycline (aTc). It also has mCherry, under the control of the pBAD promoter, meaning L-arabinose will induce mCherry transcription. The mCherry transcript has the RBS BBa_K4811000 incorporated, meaning that translation of mCherry should be inhibited by the TMS (GFP). With a GFP aptamer incorporated in the D-loop, Paul et al. (2020) showed GFP interfering with this inhibition. Higher levels of GFP lead to more mCherry fluorescence.

A schematic of the system to be tested can be seen below:

GFP is induced by aTc, mCherry is induced by L-ara, and the TMS is induced by IPTG. The hypothesis is that the TMS is able to repress translation of mCherry, by binding to the RBS, and that GFP is able to bind to the TMS, thus, releasing the TMS from the mCherry RBS, allowing translation to begin.

First, the system was tested by induction with varying levels of L-arabinose and IPTG, to find the optimal induction conditions. The results of this can be seen below:

Results_TMS(GFP), mCherry, IPTG
mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of IPTG concentration. All measurements were with 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with no inducers present (uninduced)
Results_TMS(GFP), mCherry, Arabinose
mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of L-arabinose concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM IPTG (IPTG), one only induced by L-arabinose (L-ara), and one with no inducers present (uninduced)

As described in Engineering, 1 mM IPTG and 0.1 % w/v L-arabinose were found to be sufficient conditions for simultaneous expression of mCherry and the TMS. Using these conditions, GFP was induced by aTc, which showed the following results:

Results_TMS(GFP), GFP
GFP fluorescence normalized relative to OD, 488 nm excitation/545 nm emission, as a function of anhydrotetracycline (aTc) concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM IPTG, one only induced by L-arabinose (L-ara), and one with no inducers present (uninduced)

The results showed that aTc induced GFP, with 0.5 μM having the highest fluorescence. We would have expected 1 μM GFP to have even higher fluorescence, however it seems to be lower, but with a much higher standard deviation associated with the data. With respect to mCherry fluorescence, we were not able to recreate the results of Paul et al. (2020), and the only statistical significance was for the measurement of 1 μM of aTc, which had higher fluorescence than the other measurements. However, the corresponding GFP fluorescence was lower for 1 μM aTc than for 0.5 μM aTc. This may be an experimental error associated with the 1 μM aTc measurement. There is also no statistical significance between the 0 μM aTc measurement and the L-ara control, with only L-ara as inducer, and no IPTG.

Results_TMS(GFP), mCherry
mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of anhydrotetracycline (aTc) concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM (IPTG), one only induced by L-arabinose (L-ara), and one with no inducers present (uninduced)

Validation of new TMSs

Using existing aptamers from the literature, new TMSs were constructed. This includes two manganese responsive TMSs and two theophylline responsive TMSs. They were characterized as the following composite parts:

All TMSs were characterized in E. coli BL21(DE3), where the TMS is induced by IPTG through the T7 polymerase of the DE3 system. The constructs were USER cloned into the high copy number pUC19 backbone for testing. A reporter, consisting of BBa_K4811003 was USER cloned into the low copy number pACYC184 backbone. This has mCherry under control of the pBAD promoter, meaning L-arabinose will induce mCherry transcription. The mCherry transcript has the RBS BBa_K4811000 incorporated, meaning that translation of mCherry should be inhibited by the TMS, if the inhibiting capabilities are conserved upon changing the D-loop.

A general schematic of the system to be tested can be seen below:

mCherry is induced by L-ara, and the TMS is induced by IPTG. The hypothesis is, that the TMS is able to repress translation of mCherry, by binding to the RBS, and that the ligand is able to bind to the TMS aptamer in the D-loop, releasing the mCherry RBS of the TMS, allowing translation to begin.

The two theophylline TMSs were designed using the paper published by Suess et al. (2004) for the aptamer sequence, and these were tested for mCherry fluorescence based on concentration of the compound theophylline. The results showed a statistically significant decrease in mCherry fluorescence dependent on theophylline concentration for TMS(theo2), but no statistical significance for TMS(theo1), which had a much higher standard deviation associated with its results. Together, this shows that it is possible to insert an aptamer sequence from literature and, with the appropriate design changes, adapt it to the TMS platform in order to create a TMS system that is responsive to the desired ligand in a dose-controllable way. Results are summarized below.

Results_TMS(Theo_combined)
mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of theophylline concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose.

Additionally, two manganese TMSs were tested, and the results are summarized in the figure below. A decrease in mCherry fluorescence can be seen in TMS(Mn1). To improve the Mn-dependent reporter translation inhibition, the principles that were successful in the design of TMS(Theo2) were applied to TMS(Mn2). This construct, however, had a less significant decrease in fluorescence than the first iteration of the engineering cycle.

Results_TMS(Mn_combined)
mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of manganese concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose.

Validation of PFOA TMSs

Applying the principles learned from the design of the above TMSs, 3 different PFOA aptamer designs were incorporated into the D-loop of the TMS structure starting from the aptamer described in Park et al. (2022), creating TMS(PFOA1) - BBa_K4811024, TMS(PFOA2) - BBa_K4811030, and TMS(PFOA3) - BBa_K4811025. These were tested in quadruplicates with varying concentration of PFOA and the results are summarized in the graph below. All PFOA TMSs showed a decrease in mCherry fluorescence based on PFOA concentration. The best TMS designed is the one produced in the second iteration of the engineering cycle. Crucially, and perhaps interestingly, the original inserted aptamer sequence from the Park et al. (2022), selected through a PCR-dependent technique such as SELEX, is a DNA sequence, but our TMS(PFOA)s seem to work despite being in RNA form.

Results_TMS(PFOA_combined)
mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of PFOA concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose

Discussion and future perspectives

As mentioned in Engineering, although contrary to what was expected from the original TMS paper, all the TMS biobricks produced in this work show a ligand-dependent inhibition of protein translation. This suggests a need to revise the mechanistic model of how the TMS structures exert their function. Overall, the produced TMSs show a concentration-dependent inhibitory activity whose specificity is conferred by the inserted aptamer sequence. Assuming that the observed correlation between PFOA and fluorescence is based upon a change in the structure of the TMS, a stabilization of the most stable inhibitory structure would explain the observed results.

As represented in the animation above, a ligand molecule binds to the D-loop of the TMS, and together the complex binds to the reporter mRNA. In this example the ligand represented is the theophylline molecule that binds to the TMS (Theo2) BBa_K4811011. Contrarily to the original TMS mechanism model, the presence of the ligand during translation leads to a reduction of fluorescence output, represented here as a dimming on the red shine. In regards to the main track of our project, represented in the diagram below, the ligand is PFOA binding to TMS (PFOA2) BBa_K4811030, and its presence stabilizes TMS binding on the mRNA RBS (BBa_K4811000, colored orange).

Revised model

Although the creation of various ligand-responsive TMSs was generally successful, it is worth noting that these behave in contradiction compared to what was initially expected. This is when only taken the work by Paul et al. (2020) into account. Thus, this might signal that reproducibility is dependent on other factors, which might have had an influence on our experimental. setup. Some variables that could have had an influence are pH, solubilized ions, and change in surface tension.

Theophylline is a weak base, so both the medium and intracellular pH might exert a slight effect across the theophylline experiments. The increase in effectiveness of the second version of the TMS compared to the first might shed light on the role of the structural features to look for during the part design. The original aptamer sequence selected contained a stem that connects the aptamer RNA sequence to the translated mRNA. This stem has the functional role of transmitting the conformational change of the aptamer upon theophylline binding to the mRNA by changing the accessibility of the RBS cassette through a one-nucleotide slipping mechanism. Although this mechanism exerts a response in vivo on the native mRNA, its function in a TMS design is unknown, and could make the TMS structure less sensitive to the ligand binding. Furthermore, its removal during the designing of TMS(Theo2) is seen to increase its sensitivity.

Manganese is a solubilized ion, which would change the osmotic pressure. One factor to be considered is that, the original aptamer inserted in the TMS structure was taken as a biobrick from the iGEM registry (BBa_K902074), and this sequence is native of E. coli, where it controls the production of the efflux pump for the export of manganese ions themselves. Hence, the intracellular concentration levels of manganese will appear broad on a population level, not only making E. coli not the best organism to characterize this structures into, but also likely leading to an organism-dependent effectiveness of the two Mn TMSs.

PFOA changes the surface tension of water, which may lead to better shaking of the culture during induction. Furthermore, association of PFOA to the bacterial cell membrane has been suggested in the literature but not deeply investigated, and possible disruption of cell membranes or membrane-associated proteins might impose additional stress conditions that should be accounted for while designing experiments.

All these factors could be explored to determine the mechanism of action of the TMS. Having said this, we managed to obtain a statistically significant, dose-dependant response for both theophylline TMSs, of which BBa_K4811011 shows the highest response, and two PFOA TMSs, with BBa_K4811030 showing the highest response. Naturally, further experimental confirmation is needed before definite conclusions about the produced TMSs can be reached. Specifically, a deeper insight into the mechanistic action of TMSs is necessary, and could aid the design of more TMSs in the future. We therefore encourage future teams to work with the TMS system to elucidate the exact method of action, as there is potential for elucidation and we have seen a wide variablity between operators. For future experiments, the addition of rigorous negative controls might be advisable in order test the specificity of the system and to eliminate contributions of false positives. Further factors to be tested might include:

  • Toxicity of the selected ligands
  • Toxicity of reporter production-associated stress
  • Expression of efflux pumps
  • Culturing conditions
  • Strain-dependency of the system
  • Functionality in other types of organisms
  • Effect of ligand-associated conformational change on TMS-mRNA interactions
  • Re-design of the aptamer insertion area onto the TMS body

We strongly believe that with the contribution and validation of the scientific community, this technology can become a valuable tool for the designing of various biosensors, as well as a bioengineering tool for the study of gene function.

You can find our raw data here: Raw data