Composite

Part:BBa_K2240000

Designed by: iGEM2017_HKUST   Group: iGEM17_Hong_Kong_HKUST   (2017-10-18)


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

This part can detect the deliberately released stimulus to initiate the process of 'knockout'. Considering 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, is the inducer that is originated from V. fischeri, a lipid molecule that can diffuse across the bacterial cell membrane to facilitate cell-cell communication.

This part begins with the TetR repressible promoter (BBa_R0040), which can act as a constitutive promoter to activate LuxR (BBa_C0062) under the absence of repressor TetR. Once 3OC6HSL is added, LuxR will form a complex with 3OC6HSL and then activate the downstream promoter, PluxR (BBa_R0062). In the end, the production of LuxR can be boosted by the accumulation of LuxR.

After the activation of PluxR, autoinducer synthetase (BBa_C0061) would synthesize 3OC6HSL, which then bind to LuxR, 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 a positive feedback loop, hence, this further induces PluxR. Apart from this, 3OC6HSL can also diffuse to the extracellular environment and induce the nearby cells.

Owing to the positive feedback loop, this part starts emitting signals whenever it receives 3OC6HSL molecules. As a result, it is expected to elevate the activation level under induction.

Considering the difficulties encountered by the previous iGEM teams, which was the leakiness of PluxR (See experience in BBa_F2620), we tried to get rid of this by adding antisense RNA binding regions and antisense RNA (asRNA), which could lower basal level. Ideally, it helps tackle this thorny issue.


Usage & Biology

The antisense RNA (asRNA) has two important characteristics which boost the efficiency in reducing leakiness: the sequence complementary to the mRNA of ABR, and a Hfq (RNA binding protein) binding site. The affiliation of asRNA to the complementary ABR can prevent ribosome from binding to the mRNA of the targeted RBS. Meanwhile, the Hfq binding site helps reduce the translation of the 3OC6HSL/LuxR complex since Hfq binding site could recruit RNase to degrade the targeted RNA chain (Hoynes-O’Connor & Moon, 2016). With these two effects, the leakiness of PluxR is lowered.

There are two asRNAs binding regions (ABR) in total. One is placed right before the targeted ribosomal binding site (RBS), which is upstream of the autoinducer synthetase for AHL (BBa_C0061) and GFP generator (BBa_E0240).

Under the presence of 3OC6HSL, 3OC6HSL/LuxR complex can repress PluxL, reducing the production of the asRNA, in turn, increasing the production of LuxI. It means that the maximum level of LuxI translation can be achieved after 3OC6HSL induction.

Based on the reasons above, it is expected that the system would become more sensitive due to the positive feedback loop, whereas the promoter leakiness could also be reduced.


Antisense RNA Type 1 & 2

To provide an alternative of antisense RNA, two antisense RNA sequences (type 1 and type 2) with a few base pairs differences were designed. Both of them were obtained from a journal and they were then slightly modified (Hoynes-O’Connor & Moon, 2016). Being incorporated into an inducible system with positive feedback loop (PFB) that can produce GFP, the efficiency in reducing the basal level can be evaluated via measuring the GFP output under the presence of AHL.

Results
Fig.1 Error bar presents SD from 6 biological replicates.
Fig.2 Error bar presents SD from 6 biological replicates.

























Constructs involved

  1. w/o PFB (without positive feedback loop): pSB1C3-BBa_T9002
  2. w/ PFB (with positive feedback loop): pSB1C3-BBa_F2620-C0261-E0240
  3. PFB + asRNA1: pSB1C3-BBa_K2240000
  4. PFB + asRNA2: pSB1C3-BBa_K2240003

Finding 1. Antisense RNA (asRNA) can reduce basal expression level

The effectiveness of basal level reduction for asRNA type I and II was compared with the control (w/PFB) prior to AHL induction ([AHL]=0M) and after the induction ([AHL]=1.00E-05 M). Unpaired t-test analysis suggested that their differences were statistically significant. Therefore, the two asRNAs could lower the basal level of the targeted promoter.

Finding 2. Antisense RNA type II reduces basal level more significantly

Based on the results obtained from the unpaired t-test calculation, there were significant differences between antisense RNA type I and type II under both conditions - before and after AHL induction (1.00E-05 M). Fig.1 and 2 implied that the construct with asRNA type II could reduce basal expression level more significantly than asRNA type I. It was conjectured that this might be caused by the GC content difference in their complementary sites (55% for asRNA type II comparing to 47.4% for asRNA type I) since the two asRNAs only differed in their GC content and sequences.


Signal Amplification

GFP expression was measured in order to examine the efficiency for the positive feedback loop to amplify the signals with the presence of AHL inducer.

Results
Fig.3 Error bar presents SD from 6 biological replicates.
Fig.4 Error bar presents SD from 6 biological replicates.





















Finding 3. Positive feedback loop works after AHL induction

There was also notable statistical differences when comparing the changes before and after induction using the two-tailed paired t-test analysis for both PFB+asRNA1 (p<0.001) and PFB+asRNA2 (p<0.01). Though the calculations are not shown here, the average of fluorescence/OD600 for PFB+asRNA2 was 4,199.18 while the average after its induction was 6,121.58, showing an increase for 1,922.4 GFP/OD600 (45.8% growth) after 3 hours. On the other hand, PFB+asRNA1 also experienced an increase by 4,765 GFP/OD600 (65.7% growth) under the same condition (Fig.4). An increase in expression after the AHL induction suggests that asRNA could work as expected.


Conclusions from the Experiments

The antisense RNAs construct can verify the following hypotheses:

1. There was a significant decrease in basal level when antisense RNA type I and II were incorporated into the system.

2. Antisense RNA type II reduced basal level more effective than antisense RNA type I.

3. The positive feedback loop was functional.



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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