Design

Overview

In order to design a cohesive experimental plan, we begun by defining the parameters of interest. This was done with the intention of finding what constructs were necessary to not only generate parameter-data to fit the model but evaluate our parts both qualitatively and, when applicable, quantitatively.

In silico indications

Through simulations of our intended construct with parameters defined a priori, we were able to draw some conclusions as to the validity of our system as a microplastic biosensor and generated some insight into the expected behavior which we could apply in the wet-lab setting. The model reflected very high dose sensitivity with regards to both ligands, which serves as an indicator that our system ought to work as a microplastic sensor. While we naturally expect lower affinity than that of the simulated natural ligands, it should still display somewhat similar characteristics. This is important as the concentrations of measured homologues will most likely be low. Thus, we can pass up on introducing positive autoregulatory motifs to our intended network topology. The rise-time of the GFP concentration suggests a slowly forming output signal, with over 10 hours before it reaches 90% of steady-state concentration (for varying concentrations of ligands). Thus, our fluorescence readings will have to be conducted first after several hours - most likely 10 hours post induction. Furthermore, improving the kinetics of a single node in our network will not particularly change the association time of GFP. That is to say, we do not need to “improve” any genes, for example through mutagenesis or simply changing some sequence to one with similar function, in an attempt to decrease the time to output signal as it will most likely not work. The IPTG concentrations will affect the overall gene transactivation and the associated GFP concentration. Thus, to achieve measureable results, we will use a relative high concentration corresponding to that of at least 1 mM.

Control construct design

In this section we introduce the constructs intended to be used as controls. The gene architecture is designed to best generate parameters to fit the model and evaluate individual gene behaviors.

The pETDuet-1 vector was chosen for all desired protein expressions in this project as recommended by our advisor Dr. Nélida Leiva Eriksson. Genes were synthesized at Eurofins Genomics and cloned into the pETDuet-1 vector using the restriction site pairs NcoI/BamHI and NdeI/AatII. The restriction sites were chosen based on their location downstream the two T7 promoters. In addition, it was required that these restriction enzymes were single site cutters which was ensured by using the RestrictionMapper online tool. All the gene sequences used were codon optimized for E. coli by using Eurofins’ and IDT’s codon optimization tools prior to ordering the constructs. Furthermore, all genes encoding a protein were chosen to be ended with the double stop codon, TAA TAA.

NahR

The control plasmid carries the nahR gene with a histidine tag, located under the T7 promoter. The purpose of this control plasmid was to see if it is possible to induce expression of NahR, using the drawn-up design illustrated in fig. 1. By utilizing the His-tag, the protein could be purified in order to determine its concentration.

The DNA sequence for the NahR protein was obtained from the BioBrick part BBa_K228004.

Figure 1: nahR sequence with His-tag located under the T7 promoter.

sfGFP

The construct with superfolder green fluorescent protein (sfGFP) located under the T7 promoter, as depicted in fig. 2, was constructed to be used as a positive reference for the fluorescence signal.

Figure 2: sfGFP located under the T7 promoter.

GFP1-9

The purpose of expressing the largest fragment of the tripartite split-GFP was to examine if the protein emit any fluorescence signal and if so, how large the fluorescence signal is compared to one from the sfGFP. A His-tag with 6 histidine residues was added to enable purification and measurements of protein concentration. Construct illustrated in fig. 3.

The DNA sequence for the tripartite split GFP was obtained from a study by Cabantous et al. (2013) [40].

Figure 3: GFP1-9 located under the T7 promoter.

NahR with sfGFP under Psal promoter

This control construct, illustrated in fig. 4 was designed in order to investigate if added salicylate increases the transcription rate of the reporter protein. Furthermore, the purpose was to examine the level of leaky expression obtained with no salicylate added. As explained in the theory section, NahR binds to and activates the Psal promoter in presence of salicylate resulting in expression of the reporter protein, in this case sfGFP.

The DNA sequences for sfGFP and Psal promoter were obtained from SnapGene’s Plasmid file and NCBI’s database, respectively.

Figure 4: nahR located under the T7 promoter, inducible by IPTG. After induced expression of the NahR protein, the latter binds to Psal and the sfGFP is expressed.

NahR with GFP1-9 under Psal promoter

Construct with NahR expressed under the T7 promoter and GFP1-9 located under the Psal promoter, as illustrated in fig. 5. The purpose of this control was to confirm the expression of GFP1-9 as a result from activation of the Psal promoter.

Figure 5: The NahR gene located under the T7 promoter, inducible by IPTG. After induced expression of the NahR protein, the latter binds to Psal promoter (in presence of salicylate) and the GFP1-9 is expressed.

GFP10-hER-𝛂 LBD-GFP11

The control was constructed to confirm expression of the fusion protein GFP10-hER-α LBD-GFP11, illustrated in fig. 6. The protein is attached to a 6 histidine residue tag to facilitate purification and consequently allow concentration measurements. The DNA sequence for the hER-α LBD was kindly provided by Dr. Huimin Zhao upon request.

Figure 6: GFP10-hER-α LBD-GFP11 located under the T7 promoter with His-tag.

GFP1-9 + GFP10-hER-α LBD-GFP11 device

Construct with GFP1-9 and GFP10-hER-α LBD-GFP11, each located under a T7 promoter. This control was designed to investigate the reassembling of the tripartite split GFP and measure its fluorescence.

Figure 7: Construct with genes encoding GFP1-9 and GFP10-hER-α LBD-GFP11 each placed under their own T7 promoter. Note that the lac operators have been omitted for clarity.

Intended experiments

Induce cell culture with different IPTG concentrations at several OD₆₀₀ values to find an optimal condition for protein expression. SDS-PAGE would be performed for rough estimation of protein amounts.
Purify His-tagged proteins - NahR (fig. 1), GFP1-9 (fig. 3) and GFP10-hER-α LBD-GFP11 (fig. 6), with Immobilized Metal ions Affinity Chromatography (IMAC) and thereafter perform protein concentration measurements using spectrophotometer.
Using fluorometer to measure fluorescence signal of sfGFP (fig. 2) and GFP1-9 (fig. 3).
Investigate the expression rate of sfGFP (fig. 4) and GFP1-9 (fig. 5) by using several concentrations of inducer salicylate as well as examine the leaky expression in absence of salicylate.
Use tamoxifen as an antagonistic ligand to investigate whether the conformational change of the GFP10-hER-α LBD-GFP11 construct happens. The conformational change causing reassembly of the tripartite split GFP would result in fluorescence signal, measured with fluorometer. The same experiment would be performed without added tamoxifen in order to see whether the conformational change is needed for the tripartite split GFP to reassemble.