<td>The cell wall/membrane can cause permeability limitations on the response time of the biosensor.</td>
</tr>
</table>
<p><b>Table 1.</b> Shows the differences between cell-based and cell-free biosensors</p>
<pstyle="text-align:center;"><b>Table 1.</b> Shows the differences between cell-based and cell-free biosensors</p>
<br>
<p>We were inspired by Edinburgh’s 2020 iGEM team (Finding NEMO) design for their cell-free transcription-only biosensor. Their design only required the transcription of fluorescent RNA aptamers which then bind to a fluorophore to produce a readable output. The biosensor only requires T7 RNA polymerase, chemical energy from adenosine trisphosphate (ATP) and NTPs to produce this readable output. From this design they were able to produce an arsenic biosensor which could detect arsenic at levels as little as 7µM and could produce a fluorescent output within 15 minutes. This short response time is caused by the design not needing ribosomal translation which is often the rate-limiting factor in biosensor design [1]. These promising results allowed us to expand upon their design so that we could detect more metals like mercury, lead and cadmium and test a variety of different fluorescent RNA aptamers. </p>
<p>We were inspired by <ahref="https://2020.igem.org/Team:Edinburgh">Edinburgh’s 2020 iGEM team (Finding NEMO)</a> design for their cell-free transcription-only biosensor. Their design only required the transcription of fluorescent RNA aptamers which then bind to a fluorophore to produce a readable output. The biosensor only requires T7 RNA polymerase, chemical energy from adenosine trisphosphate (ATP) and NTPs to produce this readable output. From this design they were able to produce an arsenic biosensor which could detect arsenic at levels as little as 7µM and could produce a fluorescent output within 15 minutes. This short response time is caused by the design not needing ribosomal translation which is often the rate-limiting factor in biosensor design [1]. These promising results allowed us to expand upon their design so that we could detect more metals like mercury, lead and cadmium and test a variety of different fluorescent RNA aptamers. </p>
<p>The transcription of the fluorescent RNA aptamer is controlled by a transcriptional repression mechanism. This is where a protein that binds to a promoter will block subsequent binding of an RNA polymerase. In our design this is induced by heavy metal transcription factor which can bind to the heavy metal transcription factor binding site downstream of the T7 promoter; this will impede the function of the T7 polymerase. However, if there is a metal ion present in the reaction then the heavy metal transcription factor will then leave the linear biosensor and bind to the metal ion, this allows the RNA aptamer to be transcribed. Once this RNA aptamer is bound to a fluorophore it will produce fluorescence, this can be used as the readable output (Figure 1). </p>
<p>For transcription factor design we found well-characterised metal sequestering operons: MerR, PbrR and ArsR [2-4]. These operons all bind our metals interest: mercury, cadmium, lead and arsenic as these are the most common pollutants in Ghana’s waste [5]. We used the sequences of these operons to find the promoter’s which were then used as the transcription factor binding sites for the linear biosensor. We then took the transcription factors genes from these operons and were assembled into an expression cassette. These expression cassettes were then transformed into competent cells. These cells could then be lysed to obtain a cell lysate containing the heavy metal transcription factor which was used for the biosensor reaction. </p>
<p>We decided to use the MerR transcription factor for mercury (<ahref="http://parts.igem.org/Part:BBa_K4390004">BBa_K4390004</a>), a mutated MerR for cadmium (<ahref="http://parts.igem.org/Part:BBa_K4390003">BBa_K4390003</a>) as it has shown better binding to cadmium than wild-type merR [6], lead binding protein for lead (<ahref="http://parts.igem.org/Part:BBa_K4390005">BBa_K4390005</a>) and arsR for arsenic (<ahref="http://parts.igem.org/Part:BBa_K4390002">BBa_K4390002</a>). All of the expression cassettes were composed of the constitutive promoter J23100 (<ahref="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a>) and a ribosome binding site (<ahref="http://parts.igem.org/Part:B0034">BBa_B0034</a>) upstream of the transcription factor and the weak synthetic L2U2H09 Terminator (<ahref="http://parts.igem.org/Part:BBa_K4390001">BBa_K4390001</a>) downstream of the promoter. The only parts we changed were the transcription factor genes depending on what we wanted to express. All the constructs were assembled using joint universal modular plasmids (JUMP) [7] and were assembled into a JUMP Level 1 vector plasmid for expression in <i>E. coli</i> TOP10.</p>
<p>We decided to use the MerR transcription factor for mercury (<ahref="http://parts.igem.org/Part:BBa_K4390004">BBa_K4390004</a>), a mutated MerR for cadmium (<ahref="http://parts.igem.org/Part:BBa_K4390003">BBa_K4390003</a>) as it has shown better binding to cadmium than wild-type MerR [6], PbrR for lead (<ahref="http://parts.igem.org/Part:BBa_K4390005">BBa_K4390005</a>) and ArsR for arsenic (<ahref="http://parts.igem.org/Part:BBa_K4390002">BBa_K4390002</a>). All of the expression cassettes were composed of the constitutive promoter J23100 (<ahref="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a>) and a ribosome binding site (<ahref="http://parts.igem.org/Part:B0034">BBa_B0034</a>) upstream of the transcription factor and the weak synthetic L2U2H09 Terminator (<ahref="http://parts.igem.org/Part:BBa_K4390001">BBa_K4390001</a>) downstream of the promoter. The only parts we changed were the transcription factor genes depending on what we wanted to express. All the constructs were assembled using joint universal modular plasmids (JUMP) [7] and were assembled into a JUMP Level 1 vector plasmid for expression in <i>E. coli</i> TOP10.</p>
<p>Our constructs contain a double-strand DNA encoding the upstream promoter which controls the transcription of the downstream signalling component, when this component is transcribed, it will activate the fluorescence. The design is driven by a T7 RNA polymerase which uses the strong T7 promoter which can then transcribe the RNA aptamer surrounded by the F30 scaffolds. </p>
<p>We designed a variety of different biosensors depending on which metal we wanted to detect, which only required changing the transcription factor binding site (See Figure 2). We also decided to use a variety of different RNA aptamers to see if there were differences in the fluorescent output when the different RNA aptamers were bound to the DFHBI fluorophore to see which produced the strongest output. We decided on using Squash, Broccoli, iSpianch and Spinach 2 as these have all been shown to bind to DFHBI and produce fluorescence [2,8]. </p>
<figcaption><b>Figure 2.</b> Shows the sequences of the of all the linear Spinach2 biosensors. a) Arsenic biosensor which contains the arsR binding site. b) Mercury and Cadmium biosensor has the pmerT promoter which both the merR and mutated merR can bind to c) Lead biosensor which uses the ppbrT promoter as the binding for the pbrR repressor.</figcaption>
<figcaption><b>Figure 2.</b> Shows the sequences of the of all the linear Spinach2 biosensors. a) Arsenic biosensor which contains the ArsR binding site. b) Mercury and Cadmium biosensor has the PmerT promoter which both the MerR and mutated MerR can bind to c) Lead biosensor which uses the PpbrA promoter as the binding for the PbrR repressor.</figcaption>
