Difference between revisions of "Part:BBa K2036024"

Latest revision as of 05:58, 25 October 2016

placm-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase

We construct this circuit to apply our prokaryotic signal filter.
As the figure shows, Clll(BBa_K2036001) can combine with Ftsh and thus competitively preventing the degradation of Cll and Cll can activate the promoter pRE.
When body intake large amount of lactose .The lactose signal as pulse signal induce the transcription of downstream components. Then cro, Cll and Clll will have a low and transient expression level , these parts form a positive feedback system and direct stable expression of Cro, Cro binds to cro binding site and blocks the pRM promoter so that only gene iLDH will be expressed and can transform lactic acid into pyruvate.
When PH is in the range of 7~9 , Cl will be expressed and bind to Cl binding site ,then block the expression of iLDH nd Clll, which will shut down positive feedback and lead to the expression of β-galactosidase and the degradation of lactose.

Fig1: Whole circuit of the application of prokaryotic signal filter.

Protein&protein reaction

We had submitted and documented RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036014) and RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag ([https://parts.igem.org/Part:BBa_K2036015 BBa_K2036015). These two parts were to test whether CIII can protect CII from being degraded by Ftsh by competitive inhibition.

Fig2: FtsH degradation test of CIII

According to the Flourescence measurement curve above, we can see clearly that GFP level of CIII test circuit increased over time and it showed significant difference from two control groups. It indicates that tandomly expressed CIII can efficiently protect CII from being degraded by Ftsh.

Protein&promoter

CII (BBa_K2036000) functions as a transcriptional activator to direct promoter RE, so we constructed CII-TT-pRE-RBS-GFP-LVAssrAtag as test group and pRE-RBS-GFPLVAssrAtag as CK to see if CII efficiently activate pRE.

Fig3: CII and pRE activation test

According to the Flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK. We also did Fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter.

Fig4: Fluorescence detection

CI is a repressor from bacteriophage lambda. To test its interaction with pR promoter, we constructed CI-TT-pR-RBS-GFPLVAssrAtag-PET-Duet-1 and take pR-RBS-GFPLVAssrAtag-PET-Duet-1 as control to test its inhibition function. As the Relative Fluorescent intensity measurement data shows, CI can inhibit pR in minor degree but the leakage expression under pR can’t be ignored, so we should consider to increase the binding sites within pR or the amount of CI coding sequence in the circuit.

Fig5: CI-pR inhibition test

We also detected GFP reporter in E.coli after induction of 20minute, 120minutes and 240minutes through 20 times of amplification (seen from the figure below). From figure we can find the fluorescence of both two groups was increasing over time and it is obvious that the test group which contains CI expressed less GFP protein than control group. The results verify the inhibition of CI to pR from a more intuitive way.

Fig6: Fluorescence detection

We characterized cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains cro expressed less GFP protein than control group over time. It proves that cro can effectively bind pRM to block its downstream gene’s transcription.

Fig7: Cro and pRM inhibition test

Tri-stable function

Ptrp2 (BBa_K1592024) is an improved part from HUST-China 2015, we employed it as one of our signal sensor to test our tri-stable switch. We constructed ptrp2-GFP-pSB1C3 to determine an appropriate induction concentration.

Fig8: According to the GFP expression curve, IAA induction of ptrp2 with 50μM final concentration is a better choice relatively.

In order to prove that our toolkit is efficient to switch two interest genes’ expression from GFP to RFP and to eliminate the accumulation of expressed protein to interfere our measurement. We fused a degradation tag at the amino terminal of our reporter. And we used plac from the Rgistery (BBa_J04500) to characterize the degradation tag LVAssrA. We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with Excitation light 495nm.

Fig9: LVAssrAtag degradation rate measurement under plac

From the figure above, we are sorry to find that plac can not be prohibited from leakage, as there are nearly no difference between the test and control group. But we are confident to prove the high degradation efficiency of the tag as more than two thirds of the GFP degraded within 90 minutes which also offered an interesting and useful tool for rapidly down regulating certain target protein.

Beta-galactosidase activity:

Due to the limited time before wiki freezing, we didn’t completed the test of lactic balance function of our engineered strain in vitro. But we tried to characterize pH-induced beta-galactosidase’s expression level to prove that half of our application circuit (BBa_K2036024) works. We tested enzyme activity of our strain cultured at pH6.5, 7.5 and 8.5.

Fig10: Beta-galactosidase activity

As the data shows, beta-galactosidase activity of our strain cultured at pH8.5 was significantly higher than the other two groups which is corresponding to our expectations: When pH comes back to 7~9, our strain will sense the change and express beta-galactosidase.

Sequence and Features