Team:Thessaloniki/Demonstrate

Demonstrate

Demonstrate

The ultimate goal of our project was the design of stabilized promoters that achieve constant gene expression regardless of their genetic context. We created a functional toolbox that enhances stability and robustness of synthetic biological systems by decoupling gene expression from copy number.

We demonstrate that our project worked as expected by achieving gene expression decoupled from copy number.

TALE Stabilized Promoters

In order to investigate the function of TALEsp1-Pupsp1 stabilized promoter (BBa_K2839000) and TALEsp2-Pupsp2 stabilized promoter ( BBa_K2839014 ) we measured their fluorescence, as well as the fluorescence of a constitutive promoter across 3 plasmids with different Origins of Replication (psc101, p15A, pUC19-derived pMB1). For the sfGFP fluorescence intensity measurements, flow cytometry was our primary measuring method, while a plate reader was also used.

Fluorescence Intensity Measurements of the Constructs were conducted into DH5a E.coli cells

Figure 1. TALE1sp1 Pupsp1, TALEsp2 Pupsp2, non stabilized constitutive pT7A1w1 promoter flow cytometry fluorescence measurements at three different copy numbers. Error bars represent standard deviation from three biological replicates.

Figure 2. TALE1sp1 Pupsp1, TALEsp2 Pupsp2, non stabilized constitutive pT7A1w1 promoter Plate Reader fluorescence measurements at three different copy numbers. Error bars represent standard deviation from three biological replicates.

The results demonstrate that sfGFP expression level, under the control of TALEsp1-Pupsp1 stabilized promoter () and TALEsp2-Pupsp2 stabilized promoter (BBa_K2839014), remains stable when expressed from vectors with different copy number. Whereas, sfGFP expression driven from a non stabilized constitutive promoter changes when different copy number plasmids are used for its expression.

Inducibility

Expanding our system, we implemented a theophylline responsive riboswitch (BBa_K2839017) to the TALE stabilized promoters and demonstrated that they can be induced to the desired expression level.

Figure 3. Theo27 response to differential induction by theophylline. Fluorescence was measured 7 hours after initial induction with theophylline.

Figure 4. Behaviour of 12.1 Theophylline riboswitch driving the expression of the same marker with different N-terminus amino-acid sequences.

Fusion of sfGFP with 33 amino-acids from the protein luciferase, allows the riboswitch to function properly and respond to different concentrations of theophylline.

FIgure 5. TALE stabilized promoter with translational control. Induction of the TALE stabilized promoter driving sfGFP expression under the regulation of the 12.1 theophylline riboswitch. (n=3).

Fluorescence Intensity Measurements of the TALEsp1 stabilized Promoter with translational Control (BBa_K2839012) were conducted into DH5a E.coli cells, using pSB1C3 vector.

The results prove that the riboswitch responds to theophylline changing it’s conformation and thus allowing translation initiation. Furthermore,it is clear that increasing the concentration of theophylline leads to higher expression levels.