Part:BBa_K2240003
AHL sensor with positive feedback loop, GFP output and antisense RNA type 2 inhibition
The function of this part is to detect the deliberately released stimulus and initiate the process of “knockout”. Considering that the released stimulus can become diluted in an environment, a positive feedback loop is included to amplify the signal. Thus, 3OC6HSL, which is a member of acyl-homoserine lactone (AHL) family, was chosen as the inducer. 3OC6HSL originally from V.fischeri is a lipid molecule which can diffuse through bacterial cell membrane, facilitating cell-to-cell communication.
The part begins with the TetR repressible promoter or ptetR (BBa_R0040), which can be treated as constitutive promoter of LuxR protein (BBa_C0062) under no repressor TetR. This segment of the biosensor works to produce an abundance of LuxR. Once the 3OC6HSL is added to the cell environment, LuxR forms a complex with 3OC6HSL and then activates downstream promoter, Lux pR (BBa_R0062).
After the activation of Lux pR promoter, LuxI protein (BBa_C0061) then expresses. LuxI is an autoinducer synthethase which catalyzes 3OC6HSL from S-adenosyl-L-methionine (SAM) in cell. The entire parts from ptetR to LuxI generates positive feedback loop, because it increases the concentration of 3OC6HSL/LuxR complex and hence induce Lux pR more strongly. The 3OC6HSL molecule can also diffuse back to extracellular environment and affect the nearby cells.
With its positive feedback loop, the part is able to convert from signal receiver into signal emitter whenever it receives 3OC6HSL molecule. Thus, it is expected to increase the efficiency of activation in targeted environment.
Previous iGEM team encountered (See experience in BBa_F2620) the leakiness of Lux pR promoter (BBa_R0062). It is a situation when there is little expression of gene even without initial activation of AHL. This time we improved by adding sequences of antisense RNA Binding regions and antisense RNA in which their interaction can counteract the activity of basal level.
First, the antisense RNA Binding Region (ABR) is placed right before our targeted Ribosomal Binding Site (RBS), which is upstream to the LuxI (BBa_C0061) and GFP (BBa_E0040). Second, the antisense RNA is placed downstream the positive feedback loop and its reporter (Figure) together with a medium strength promoter, Lux pL (BBa_R0063). Lux pL is repressible in the presence of 3OC6HSL/LuxR complex.
The antisense RNA we use has two important characteristics that will help reduce leakiness. Firstly, it comprises of sequence that is complementary to the sequence of mRNA of ABR. The affiliation of antisense RNA to the complementary ABR prevents ribosome from binding to the mRNA of the targeted RBS. Secondly, it has a Hfq binding site, which is suspected to recruit RNase to degrade the targeted RNA chain. (Wagner, 2009)(Hoynes-O’Connor & Moon, 2016). In consequence, translation of LuxI:GFP mRNA will be reduced, so does the leakiness of Lux pR.
In the presence of 3OC6HSL, 3OC6HSL/LuxR complex will repress Lux pL. Thus, more production of mRNA of LuxI is expected. That means this system will not affect the maximum level of translation after 3OC6HSL induction.
In conclusion, it is expected that the ability of sensing in overall population will become more sensitive due to the positive feedback loop while there will be a reduced leakiness that will not affect the normal feedback activity of AHL.
We have designed 2 types of Antisense RNA in case that one of them did not work. Here is the type 2 construct.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1752
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1004
Illegal BsaI.rc site found at 2457
Illegal BsaI.rc site found at 2676
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