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 is not only activated by the regulator [http://parts.igem.org/Part:BBa_C0179:Experience LasR (BBa_C0179)], but 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_I14017:Experience Experience] - Promoter (RhlR/C4-HSL regulated), BBa_I14017: the amount of regulator [http://parts.igem.org/Part:BBa_C0171:Experience RhlR (C0171)] had an influence on the promoter's basal expression level 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 Characterized Parts

1. [http://parts.igem.org/Part:BBa_K1541009 Main Page] - sfGFP under riboregulated promoter (RhlR/C4-HSL, RR12), BBa_K1541009: this riboregulated promoter construct contains the quorum sensing promoter [http://parts.igem.org/Part:BBa_I14017 BBa_I14017] which can be activated in presence of [http://parts.igem.org/Part:BBa_C0171:Experience RhlR (BBa_C0171)] and the homoserine lactone C4-HSL, the product of the enzyme reaction catalyzed by [http://parts.igem.org/Part:BBa_C0170 RhlI (BBa_C0170)], succeeded by the gene for sfGFP. However, the promoter [http://parts.igem.org/Part:BBa_I14017 BBa_I14017] without riboregulation shows leakiness. Together with the ribogegulator 12^[18] the leakiness could be reduced.
   
   
2. [http://parts.igem.org/Part:BBa_K1541017 Main Page] - rhlI with optimized RBS, BBa_K1541017: this part is can be compared 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.