Part:BBa_K2240000

AHL sensor with positive feedback loop, GFP output and antisense RNA type 1 inhibition

The function of this part is to detect the deliberately released stimulus so as to initiate the process of 'knockout'. Considering that the released stimulus can be diluted in an environment, a positive feedback loop is introduced to amplify the signal. 3OC6HSL, which is a member of acyl-homoserine lactone (AHL) family, would be the inducer. 3OC6HSL originated from V. fischeri is a lipid molecule that can diffuse through bacterial cell membrane to facilitate the cell-to-cell communication.

This part begins with the TetR repressible promoter (BBa_R0040), which can act as a constitutive promoter of the downstream protein - LuxR (BBa_C0062) under the absence of repressor TetR. Once the 3OC6HSL is added, LuxR will form a complex with 3OC6HSL and then activates the downstream promoter, P_LuxR (BBa_R0062). In the end, the production of the LuxR can be boost, thus accumulate the LuxR.

After the activation of P_LuxR, LuxI protein (BBa_C0061), which is an autoinducer synthetase catalyzes 3OC6HSL from S-adenosyl-L-methionine (SAM) in cell. Due to the increase of the 3OC6HSL/LuxR complex, the entire part starting from P_tetR to LuxI generates positive feedback loop, hence further induce the P_LuxR. Apart from this, the 3OC6HSL molecule can also diffuse out to the extracellular environment and induce the cells nearby.

Owing to the positive feedback loop, this part would start contributing to signal emission whenever it receives 3OC6HSL molecules. As a result, it is expected to elevate the efficiency of activation under specific condition.

Considering the difficulties that previous iGEM team encountered - the leakiness of P_LuxR (See experience in BBa_F2620), we tried to get rid of this by adding sequences of antisense RNA Binding regions and antisense RNA in which their interaction could counteract the activity of basal level. Ideally, this could help tackling the thorny issue which would happen when there is little expression of gene, especially without the initial activation of AHL.

Designs & Hypothesis

The antisense RNA we used had two important characteristics which might help reduce leakiness: possessing the sequence complementary to the of mRNA of ABR, and a Hfq binding site. The affiliation of antisense RNA to the complementary ABR would prevent ribosome from binding to the mRNA of the targeted RBS. Meanwhile, the presence of a Hfq binding site would help reduce the translation of the 3OC6HSL/LuxR complex since Hfq binding site was suspected to recruit RNase to degrade the targeted RNA chain. With these two effects, the leakiness of P_LuxR could be lowered.

There were two antisense RNA binding regions (ABR) in total. One was placed right before the targeted ribosomal binding site (RBS), which was upstream to the LuxI (BBa_C0061) and GFP (BBa_E0040) while another one was placed downstream of the positive feedback loop.

Despite the action of the antisense RNA, 3OC6HSL/LuxR complex could still repress P_LuxL, increasing the production of mRNA of LuxI under the presence of 3OC6HSL. This meant that the maximum level of LuxI translation could be maintained after 3OC6HSL induction.

Based on the above reasons, it was expected that the ability in sensing the overall population would become more sensitive due to the positive feedback loop while leakiness could be reduced without affecting the normal feedback activity triggered by AHL.

Results

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1751
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1004
  Illegal BsaI.rc site found at 2455
  Illegal BsaI.rc site found at 2674