Composite

Part:BBa_K2240000

Designed by: iGEM2017_HKUST   Group: iGEM17_Hong_Kong_HKUST   (2017-10-18)
Revision as of 10:11, 27 October 2017 by Karencheng (Talk | contribs)


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, PluxR (BBa_R0062). In the end, the production of the LuxR can be boost, thus accumulate the LuxR.

After the activation of PluxR, 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 PtetR to LuxI generates positive feedback loop, hence further induce the PluxR. 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 PluxR (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.


Usage & Biology

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 (RNA binding protein) 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 PluxR could be lowered.

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

Despite the action of the antisense RNA, 3OC6HSL/LuxR complex could still repress PluxL, 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 is expected that the ability in sensing the overall population would become more sensitive due to the positive feedback loop while leakiness could be reduced at the same time without hampering the normal feedback activity triggered by AHL.


Antisense RNA

To provide an alternative, two antisense RNA sequences (type 1 and type 2) with a few base pairs different were designed for degrading the mRNA. Both of them were obtained from the paper, Development of design rules for reliable antisense RNA behavior in E. coli, and then slightly modified. Being incorporated into an inducible system with GFP expression, they were evaluated for their degradation efficiency via measuring the GFP output with the presence of AHL.

Results


AHL Sensor with Positive Feedback Loop

GFP was measured in order to examine the effectiveness of the antisense RNA in reducing the leakiness of the system after being induced by different concentrations of 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


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