Results of Cumate Switch Regulation System

We cloned the CymR-SV5 tag transcriptional unit under the constitutive promoter J23100 (BBa_K875003).




The production of the protein CymR was verified through the Western Blot analysis (here below), using an anti-SV5 antibody. Here we can see one band of almost 25.7 KDa, which corresponds to the molecular weight of the protein CymR-SV5 tag.



The J23100-CymR-B0015 was then cloned upstream the T5 promoter-cumate operator (BBa_K875001) + GFP (I13504) .
In order to verify the functionality of the system we test it using different concentrations of p-cumate. The cumate binds the CymR preventing its repression thus allowing the GFP expression.


In liquid assay:

We inoculated a 20ml culture. After overnight growth, we diluted the culture to O.D. ₆₀₀ = 0.2. Then we aliquoted 200 μl in 8 replicates in a microtiter plate at different concentrations of cumate. The reading was performed in a monochromator at 485-510nm. 

Both graphs show the relation between p-cumate concentration and the GFP fluorescence intensity at different times. In the first one, the modulation range is explored and it has been relevated that the maximum yield is reached at 30uM. In the second one the fluorescence of a culture is followed in the time and it has been noticed an interesting difference between the induced and not induced cultures. The data were normalized over the background given by the LB media.

In plate assay: 

We streaked the culture on LB plates containing different cumate concentrations.


We proved that the combination of CymR and T5 Cumate Operator creates a strong, very stringent system for protein expression with a rapid reactivity.

The goal will be to validate the activity of this system as gene guard. The limiting point could be infact the poor expression of CymR that will be put in the bacterial genome in just two copies. Future work will confirm if two copies are sufficient for repressing the expression of the T5CumateOperator-toxin carried by the plasmid.

Suicide System

Our first idea was to use the LL 37, the only cathelicidin-derived antimicrobial peptide found in humans. It was known that the LL 37 kills the bacteria, so we decided to use it as the toxin in our system.


As you can see in the pictures above this approach was unsuccessfully, so we thought that maybe LL 37 can not kill the bacteria if expressed inside them. So, we decided to use the LL 37 in another way, combining it with the T4holin from the Registry(BBa_K112000). The T4holin acts on the inner membrane, creating pores through which the LL 37 could reach the outer membrane and cause lysis.
This approach should be successful, but the T4Holin has the restriction sites (RFC 21) not compatible with the standard ones (RFC 10), so we had to transform the biobricks we needed into BglBricks according to the RFC 21.

The rational of this construct is represented below:



Meanwhile, we took an optional way as a suicide system, using the Tse2, a toxin from the registry (BBa_K314200). We cloned it downstream the T5Lac Operator Promoter and we tested it inducing and non-inducing the cells with IPTG.

The rational of this construct is represented below:



The M15 cells were transformed with our construct. Two positive colonies (colony n.11 and colony n.47), checked by PCR, were plated on +/-IPTG plates. The colony n.47 lives in the plate with -IPTG and dies in the +IPTG plate as expected.

Antibody

We produced an engeneered antibody (in different formats: SIP and scFv) using two different expression systems. In order to obtain a secreted version of the antibody, we cloned it under PelB leader sequence. Then, we fused the antibody in frame with LPP-OmaA fragment in order to display it extracellularly. All constructs were cloned downstream T5 Lac Operator.

We tested the expression of our antibodies by Western blotting.

a) T5LacO-LPP-OmpA-scFv

The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 encoding for the Lac Repressor. The recombinant bacterial culture was induced at O.D.= 0.4 (2x108 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial culture were centrifuged and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonicated and boiled for 5 min. 10μl of lysates of induced, non-induced and non trasformed bacterial cultures were resolved on SDS-PAGE. The expression of fusion protein LPP-OmpA-scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies

Expression of scFv 54.6 cloned in fusion with the LPP-OmpA leader sequence . Western blots of lysates of E.coli W3110 bacterial strain expressing the recombinant protein scFv 54.6 (45,19KDa), induced or non-induced with IPTG. The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.

OmpA-scFv fragment was cloned under Constitutive operator (BBa_J23100) and then tested in E.coli strain DH5alpha. The recombinant bacterial culture was inoculated over night at 37°C in shaker. 2ml of bacterial culture were centrifuged and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonicated and boiled for 5 min. 10μl of lysates of induced, non-induced and non trasformed bacterial cultures were resolved on SDS-PAGE. The expression of fusion protein LPP-OmpA-scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies.

Expression of scFv 54.6 cloned in fusion with the LPP-OmpA leader sequence . Western blots of lysates(C= with Lpp-OmpA-scFv 54.6; WC= without LPP-OmpA-scFv 54.6) of E.coli DH5alpha bacterial strain expressing the recombinant protein scFv 54.6 (45,19KDa) . The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.

The localization of the scFv 54.6 fused with LPP-OmpA into the outer bacterial membrane was proved by performing an immunoflorescence assay on the bacterial strain DH5alpha expressing this construct. 5x108 recombinant bacterial cells were incubated with 5μl of Mouse Anti-6HIS (0,5μg/μl) for 3hs at 4°C followed by an incubation with TRITC conjugated anti-mouse IgG .

b) T5LacO-LPP-OmpA-SIP

The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 encoding for the Lac Repressor. The recombinant bacterial culture was induced at O.D.= 0.4 (2x108 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial culture were centrifuged and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonicated and boiled for 5 min. 10μl of lysates of induced, non-induced and non trasformed bacterial cultures were resolved on SDS-PAGE. The expression of fusion protein LPP-OmpA-scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies.

Expression of SIP 54.6 cloned in fusion with the LPP-OmpA leader sequence. Western blots of lysates of E.coli W3110 bacterial strain expressing the recombinant protein SIP 54.6 (60KDa), induced or non-induced with IPTG. The blot was reacted with the Affinity purified antibody goat anti-Human IgA(α).

c) T5LacO-PelB-scFv

The construct was tested in E.coli W3110 strain which was previously transformed with p-REP 4 encoding for the Lac Repressor. The recombinant bacterial colonies were induced at O.D.= 0.4 (2x108 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial colture were centrifuged and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonicated and boiled for 5 min. 10μl of lysates of induced, non-induced and non-trasformed bacterial cultures were resolved on SDS-PAGE. The expression of fusion protein scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies

Expression of scFv 54.6 cloned in fusion with the pelB leader sequence. Western blots of lysates (C= with pelB-scFv 54.6; WC= without pelB-scFv 54.6) of E.coli HB2151 bacterial strain expressing the recombinant protein scFv 54.6 (29,25KDa). The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.

d) T5LacO-PelB-SIP

The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 encoding for the T5 Lac Repressor. The recombinant bacterial colonies were induced at O.D.= 0.4 (2x108 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial colture were centrifuged and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonicated and boiled for 5 min. 10μl of lysates of induced, non-induced and non trasformed bacterial cultures were resolved on SDS-PAGE. The expression of protein SIP 54.6-His was tested by Western blotting with anti-6HIS antibodies.

Expression of SIP 54.6 cloned in fusion with the pelB leader sequence. Western blots of lysates (SN=supernatant; P=pellet) of E.coli W3110 bacterial strain expressing the recombinant protein SIP 54.6, induced or non-induced with IPTG. The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.