Gene Circuit and Parts

Used and Characterized Pre-Existing Parts

1. [http://parts.igem.org/Part:BBa_R0062:Experience Experience] - Promoter (LuxR/3OC6-HSL regulated), BBa_R0062: the promoter's EC₅₀ could be shown to be dependent on the amount of regulator [http://parts.igem.org/Part:BBa_C0062:Experience LuxR (BBa_C0062)] in the system by using the three different constitutive promoters [http://parts.igem.org/Part:BBa_J23100:Experience BBa_J23100], [http://parts.igem.org/Part:BBa_J23109:Experience BBa_J23109], and [http://parts.igem.org/Part:BBa_J23111:Experience BBa_J23111] for the production of LuxR. The amount of regulator also demonstrated an influence on the promoter's basal expression or leakiness. Additionally, it could be determined that the promoter can also be activated by the regulators [http://parts.igem.org/Part:BBa_C0179:Experience LasR (BBa_C0179)] and [http://parts.igem.org/Part:BBa_C0171:Experience RhlR (C0171)] thus showing crosstalk with these two quorum sensing systems.
   
   
2. [http://parts.igem.org/Part:BBa_R0079:Experience Experience] - Promoter (LasR/3OC12-HSL regulated), BBa_R0079: it could be shown that this promoter, which is usually activated by the regulator [http://parts.igem.org/Part:BBa_C0179:Experience LasR (BBa_C0179)] can also be activated by the regulators [http://parts.igem.org/Part:BBa_C0062:Experience LuxR (BBa_C0062)] and [http://parts.igem.org/Part:BBa_C0171:Experience RhlR (C0171)]. Therefore, it shows crosstalk with these two quorum sensing systems.
   
   
3. [http://parts.igem.org/Part:BBa_R0071:Experience Experience] - Promoter (RhlR/C4-HSL regulated), BBa_R0071: the amount of regulator [http://parts.igem.org/Part:BBa_C0171:Experience RhlR (C0171)] demonstrated an influence on the promoter's basal expression or leakiness as well as on the steepness of the response. This was investigated using two different RBSes controlling the expression level of RhlR, i.e. the RBS [http://parts.igem.org/Part:BBa_B0034 B0034] and a RBS optimized for rhlR using the [http://salis.psu.edu/software/RBSLibraryCalculatorSearchMode Salis Lab RBS Calculator]^[13].
   
   
4. [http://parts.igem.org/Part:BBa_C0062:Experience Experience] - Regulator LuxR, BBa_R0062: it could be shown, that the regulator LuxR not only activates the promoter [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] together with 3CO6-HSL, the product of the enzyme [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)], but also with 3CO12-HSL ([http://parts.igem.org/Part:BBa_C0178 LasI (BBa_C0178)] product) and C4-HSL ([http://parts.igem.org/Part:BBa_C0170 RhlI (BBa_C0170)] product).
   
   
5. [http://parts.igem.org/Part:BBa_C0179:Experience Experience] - Regulator LasR, BBa_C0179: it could be shown, that the regulator LasR activates the promoter [http://parts.igem.org/Part:BBa_R0079 BBa_R0079] mainly together with 3CO12-HSL, the product of the enzyme [http://parts.igem.org/Part:BBa_C0178 LasI (BBa_C0178)] and only to a minor extend with 3CO6-HSL ([http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] product) and C4-HSL ([http://parts.igem.org/Part:BBa_C0170 RhlI (BBa_C0170)] product).
   
   
6. [http://parts.igem.org/Part:BBa_C0171:Experience Experience] - Regulator RhlR, BBa_C0171: it could be shown, that the regulator RhlR not only activates the promoter [http://parts.igem.org/Part:BBa_R0071 BBa_R0071] together with C4-HSL, the product of the enzyme [http://parts.igem.org/Part:BBa_C0170 RhlI (BBa_C0170)], but also with 3CO6-HSL ([http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] product) and 3CO12-HSL ([http://parts.igem.org/Part:BBa_C0178 LasI (BBa_C0178)] product), while the ladder is acting least activating among those three AHL molecules.
   
   
7. [http://parts.igem.org/Part:BBa_J23100:Experience Experience] - Promoter, BBa_J23100: the constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100] was compared to the promoters [http://parts.igem.org/Part:BBa_J23109 BBa_J23109] and [http://parts.igem.org/Part:BBa_J23111 BBa_J23111] by placing each in a construct in front of the quorum sensing regulator [http://parts.igem.org/Part:BBa_C0062:Experience LuxR (BBa_C0062)]. The system behaved as expected, thus confirming an increasing promoter strength from [http://parts.igem.org/Part:BBa_J23109 BBa_J23109] over [http://parts.igem.org/Part:BBa_J23111 BBa_J23111] to [http://parts.igem.org/Part:BBa_J23100 BBa_J23100].
   
   
8. [http://parts.igem.org/Part:BBa_J23111:Experience Experience] - Promoter, BBa_J23111: the constitutive promoter [http://parts.igem.org/Part:BBa_J23111 BBa_J23111] was compared to the promoters [http://parts.igem.org/Part:BBa_J23109 BBa_J23109] and [http://parts.igem.org/Part:BBa_J23100 BBa_J23100] by placing each in a construct in front of the quorum sensing regulator [http://parts.igem.org/Part:BBa_C0062:Experience LuxR (BBa_C0062)]. The system behaved as expected, thus confirming an increasing promoter strength from [http://parts.igem.org/Part:BBa_J23109 BBa_J23109] over [http://parts.igem.org/Part:BBa_J23111 BBa_J23111] to [http://parts.igem.org/Part:BBa_J23100 BBa_J23100].
   
   
9. [http://parts.igem.org/Part:BBa_J23109:Experience Experience] - Promoter, BBa_J23109: the constitutive promoter [http://parts.igem.org/Part:BBa_J23109 BBa_J23109] was compared to the promoters [http://parts.igem.org/Part:BBa_J23100 BBa_J23100] and [http://parts.igem.org/Part:BBa_J23111 BBa_J23111] by placing each in a construct in front of the quorum sensing regulator [http://parts.igem.org/Part:BBa_C0062:Experience LuxR (BBa_C0062)]. The system behaved as expected, thus confirming an increasing promoter strength from [http://parts.igem.org/Part:BBa_J23109 BBa_J23109] over [http://parts.igem.org/Part:BBa_J23111 BBa_J23111] to [http://parts.igem.org/Part:BBa_J23100 BBa_J23100].
   
   
10. [http://parts.igem.org/Part:BBa_C0161:Experience Experience] - Autoinducer synthetase for the AHL molecule 3OC6-HSL, BBa_C0161: by placing an alginate bead into a millifluidic chip containing sender cells, which are constitutively expressing [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] in vicinity to a bead containing receiver cells with a construct producing sfGFP in presence of 3OC6-HSL it could be shown that signal transduction was successful. Thus it could be confirmed that the synthetase is producing a quorum sensing molecule.

Our Favorite New Parts

1. [http://parts.igem.org/Part:BBa_K1541000 Main Page] - Riboregulated Promoter (LuxR/3OC6-HSL, RR12y), BBa_K1541000: this riboregulated promoter construct contains the quorum sensing promoter [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] which can be activated in presence of [http://parts.igem.org/Part:BBa_C0062:Experience LuxR (BBa_C0062)] and 3OC6-HSL, which is the product of the enzyme [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)]. However, the original promoter by itself shows high leakiness. Together with the ribogegulator 12y^[18] the leakiness could be reduced by almost two orders of magnitude and the ON/OFF ratio increased approximately 6 times.
   
   
2. [http://parts.igem.org/Part:BBa_K1541006 Main Page] - sfGFP under Riboregulated Promoter (LuxR/3OC6-HSL, RR12y), BBa_K1541006: this part contains the same riboregulated promoter system as the part [http://parts.igem.org/Part:BBa_K1541000 BBa_K1541000] succeeded by the gene for sfGFP. This construct was used to characterize the properties of the riboregulated quorum sensing promoter [http://parts.igem.org/Part:BBa_K1541000 BBa_K1541000] and can be used in a 3OC6-HSL sensor where low leakiness is desirable.

Characterized New Parts

1. [http://parts.igem.org/Part:BBa_K1541003 Main Page] - Riboregulated Promoter (RhlR/C4-HSL, RR12), BBa_K1541003: this riboregulated promoter construct contains the quorum sensing promoter [http://parts.igem.org/Part:BBa_R0071 BBa_R0071] which can be activated in presence of [http://parts.igem.org/Part:BBa_C0171:Experience RhlR (BBa_C0171)] and C4-HSL (the product of the enzyme [http://parts.igem.org/Part:BBa_C0170 RhlI (BBa_C0170)]). However, this promoter by itself shows some leakiness. Together with the ribogegulator 12^[18] the leakiness could be reduced.
   
   
2. [http://parts.igem.org/Part:BBa_K1541009 Main Page] - sfGFP under Riboregulated Promoter (RhlR/C4-HSL, RR12), BBa_K1541009: this part contains the same riboregulated promoter system as the part [http://parts.igem.org/Part:BBa_K1541009 BBa_K1541009] succeeded by the gene for sfGFP. This construct was used to characterize the properties of the riboregulated quorum sensing promoter [http://parts.igem.org/Part:BBa_K1541009 BBa_K1541009] and can be used in a C4-HSL sensor where low leakiness is desirable.
   
   
3. [http://parts.igem.org/Part:BBa_K1541017 Main Page] - rhlI with optimized RBS, BBa_K1541017: this part is similar to the part [http://parts.igem.org/Part:BBa_I9026 BBa_I9026], but contains instead of the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034] a RBS which was specifically optimized for [http://parts.igem.org/Part:BBa_C0170 rhlI (C0170)] using the [http://salis.psu.edu/software/RBSLibraryCalculatorSearchMode Salis Lab RBS Calculator]^[13]. With higher levels of RhlI, the production of the enzyme's product C4-HSL can be improved.