Team:OUC-China/Results
1. Deciding how to incubate the bacteria for the Plate-reader experiment
Before we start formal experiments, we want to know how to incubate our engineered bacteria to ensure that they perform at their best during the plate-reader experiment. When the overnight culture was diluted and then re-grown to OD600=0.6-0.8, we adopted three different methods of incubation including shock culture in a flask and EP tubes, sampling every half hour for fluorescence detection, shock culture directly on a 96-well plate with vibrating plate device, and continuous sample measurement.
As the result shows, we found 96-well plate will not affect the expression of our bacteria and finally chose 96 well plate as the most important way.
2. Verifying the function of Basic Circuits
The basic circuit is composed of a constitutively expressed allosteric Transcription Factor（aTF）a corresponding inducible promoter, and a reporter gene (see more details on Design page).
2.1 tetR-T7(tetO)-3WJdB, tetR-T7(tetO)-sfGFP
TetR-T7(tetO)-3WJdB showed recognizable response to different concentrations of tetracycline, which means this WCB is able to detect tetracycline as low as 0.01μg/ml. Moreover, the result shows 3WJdB enable the WCB to have a faster response, as the fluorescence intensity matched the concentration gradient well only after 2 hours.
The fluorescence intensity decreased with the increase of tetracycline concentration, contrary to our expectation. Moreover, it shows max fluorescence intensity with the absence of tetracycline, which means huge leakage of output signal which may be caused by the large leakage rate of T7 promoter. Since tetracycline inhibits protein production, sfGFP is also difficult to translate and thus decreases with increasing concentrations.
To further characterize these two circuits, we invited our partnership team ZJUT （see more in partnernership page）to test them in a cell-free system.
Here are the analysis and speculation of this phenomenon: There exists the phenomenon that the gradient dependent fluorescence trend may be different (usually opposite) within different concentration range. We can see from the graph below that the trend in the second testing point (1 ng/µl) has already opposite to our expectation. One possible speculation is that the cell free system is ultra-sensitive, and 1 ng/µl of antibiotic has already exceeded the operating range. We can also see that the fluorescence intensity corresponding to 0ng/µl antibiotic is also 0. This fact indicated that the basal leakage of our genetic circuit is pretty good in cell free system. In the future, we would also like to try cell free system to achieve our goal for it have better basal leakage rate and other adorable attributes such as controllability and safety. And to further prove the concept, we have to design more testing point with lower antibiotic concentration range (at least below 1 ng/µl according to the data ZJUT provided)
2.2 ctcS- T7(ctcO)-3WJdB
CtcS- T7(ctcO)-3WJdB can also quickly detect different concentrations of chlortetracycline. However, the fluorescence reached the maximum at the concentration of 1μg/ml, then gradually decreased, which shows the potential operating range of this circuit is between 0.05-1μg/ml.
2.3 mphR-ermC- T7(mphO)-3WJdB, mphR-ermC-T7(mphO)-sfGFP
Unfortunately, mphR-ermC- T7(mphO)-3WJdB showed no erythromycin - induced effects at all. The downward trend was caused by the growth of OD600 and constant fluorescent intensity. We changed different promoters in this circuit to find the reason of of no response on later experiments.
There was still no significant difference between the experimental groups.
3. Verify the function of Antibiotic resistance gene
We linked antibiotic resistance genes into the Basic circuit by molecular cloning. TetM was assembled downstream of ctcS or tetR and controlled by the same constitutive promoter (Pc2). Successful colonies were verified by colony PCR then confirmed with sequencing.
3.1 tetR-tetM- T7(tetO)-3WJdB
TetM gene confers antibiotic resistance by producing ribosomal protection proteins, as an result, the WCB will have a wider operating range and keep robust when detecting relatively high concentration of antibiotics. The engineered bacteria were incubated in tetracycline at a maximum of 50 μ g/mL, and still maintained high fluorescence after 4 hours, indirectly reflecting the protective effect of tetM.
3.2 ctcS-tetM- T7(ctcO)-sfGFP
With the insertion of tetM gene, the reversed induction effect of ctcS-tetM-T7 (tetO)-sfGFP was corrected. In the absence of tetM, the inhibition of tetracycline or chlortetracycline on protein production is more significant than its induction. However, in the presence of tetM, the inhibition of sfGFP translation was reduced, and the normal induction effect become dominant.
4. Verify the construction of ‘improved circuit’
To construct the ‘improved circuit’ the KB2 and sgRNA are linked to both sides of the ‘basic circuit’. Successful colonies were verified by colony PCR then confirmed with sequencing.
5. Verify the strand replacement reaction between KB2 and 3WJdB
To repress the leakage of 3WJdB, KB2 (a small antisense RNA which can bind to 3WJdB) is inserted downstream of a constitutive promoter (Pc1). By tuning the strength of Pc1, the output signal can be regulated
The result of this experiment proved that it’s valid to repress the fluorescence in vivo, which strongly support the feasibility of the ‘improved circuit’.
6. Verify CRISPRi
We constructed a testing circuit to verify the CRISPRi in our WCB. An sfGFP gene was set downstream of the sgRNA binding site (bs2). The expression of dCas9 on another plasmid can be induced by 3OC6HSL (a kind of quorum sensing signal molecule) with a Plux promoter. The result shows that the fluorescence decreased with the increasing concentration of 3OC6HSL. It was proved that CRISPRi works properly.
From now on, the output signal suppression and de-suppression functions of the ‘improved Circuit’ in our whole project have been verified respectively.