Three key elements contributed to MetaSense’s design as a more efficient, accurate, user-friendly biosensor:
• A two-plasmid architecture allowed for greater control regarding the expression of multiple proteins, contributing to more regulatable sensitivity in our activator/repressor systems.
• Utilizing the benefits of cell-free protein synthesis, our paper-based sensors allowed for ease of storage, transportation, and citizen usage, while significantly decreasing the bio-contamination risks and lack of precision of common whole-cell biosensors.
• With sfGFP as the reporter gene, results of the paper test could be easily interpreted and quantified with limited scientific machinery. One of our next steps, replacing sfGFP with various colorimetric outputs, would allow for citizens to visually see the quantified test results, eliminating the need for any laboratory equipment at all.
The specific circuitry of our three two-plasmid systems are outlined below:
LuxR (BBa_K2806004) and pLux (BBa_K2806005)
Before delving into the construction of the lead and chromium-sensing plasmids, the team chose to model our system using LuxR and pLux. They are well-documented and studied proteins, especially in the context of iGEM. Thus, investigating their utility cell-free to produce sfGFP, we thought, would provide telling indication of the potential of other activator or repressor systems.
On the first plasmid, LuxR is constitutively produced, and in the presence of AHL, is able to bind to the pLux promoter, initiating production of sfGFP. Initial cell-free experiments examining generated fluorescence over time with LuxR/pLux were promising, but indicated a need for a greater signal output.
To achieve this, the original pTet promoter on the first plasmid was replaced with T7 (as shown) - the latter represents a stronger, unregulated, processive polymerase, which is important to us because we want LuxR to be present in excess for accurate sensing. For this same reason, among others, the T7 promoter couldn’t be used on the second plasmid so as to avoid a ‘leaky’ system.
Additionally, a PHP Stability Hairpin was added to the second plasmid, which aids in preventing exonuclease activity and increasing the longevity of the RNA.
As seen on our Results Page, these improvements led to a stronger fluorescence signal; in the future, and as is seen below, the LuxR gene and pLux promoter will be replaced with chromium and lead-specific genes. The modified plasmid backbones, however, will remain the same.
Chromium Repressor (BBa_K2806000) and Chromium Promoter (BBa_K2806001):
In our ideal chromium-detecting two-plasmid system, the T7 promoter would constitutively produce ChrB, which binds to the ChrP promoter on the second plasmid. This binding prevents the production of sfGFP. However, when hexavalent chromium is present, it binds to the repressing ChrB protein, and transcription and translation of sfGFP can occur, generating a fluorescent signal.
Lead Repressor (BBa_K2806002) and Lead Promoter (BBa_K2806003):
Similarly, in our ideal lead-detecting two-plasmid system, the T7 promoter would constitutively produce PbrR, which binds to the PbrAP promoter on the second plasmid, preventing the production of sfGFP. However, when lead ions are present, the toxic metal binds to the repressing PbrR protein, and transcription and translation of sfGFP can occur, generating a fluorescent signal.
