Team:Thessaloniki/Results

Result

Result

Overview

This year, we aimed to create a toolbox of stabilized promoter systems that allows for constant gene expression, independent of gene/plasmid copy number. In order to achieve that, we designed two different stabilization systems, utilizing TAL Effectors and CRISPR interference. To allow for control over gene expression, we introduced a theophylline riboswitch that, when induced with different concentrations of theophylline, achieves further control over stabilized gene expression at the desired level. This page contains all the results we obtained, the conclusions we arrived at, the debugging we performed, as well as our future plans.

All measurements were performed in biological replicates (n=3), as a chassis we used DH5a E. coli cells, maintained at mid-log phase and as reporter, we measured sfGFP fluorescence intensity (ex. 488nm).

All relevant protocols can be found at our protocols page

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.

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. Flow cytometry and plate reader data both point towards the decoupling of sfGFP expression from the plasmid copy number. Unexpected behaviour was seemingly observed by the constitutive promoter in the flow cytometry assay. A possible cause might be cross-sample contamination.

CRISPRi stabilized promoter

In order to investigate the function of the CRISPRi stabilized promoter (BBa_K2839016), we measured its fluorescence outut, as well as a constitutive promoter’s fluorescence across 2 plasmids with different Origins of Replication (psc101, pUC19-derived pMB1). dCas9 driven by the PTet promoter (BBa_R0040), is expressed from a pSB3K3 vector (p15A ori), co-transformed in the cells measured.

In order to prevent any growth rate reduction effects due to the over-expression of dCas9 and ensure that it is expressed at a high enough level so as to allow a wide range of copy number independence, the Optical Density at 600nm of cells expressing dCas9 at different inducer (doxycycline) concentrations was plotted over the concentration used 6 hours after induction (Figure 3). The desired level of expression was determined to be achieved between 0.8 and 1.2 ng/ml of inducer. These 2 concentrations are later used to induce the expression of dCas9 in all experiments.

Figure 3. Cell growth inhibition caused by different expression levels of dCas9, induced by different concentrations of doxycycline.

From figure 3, it is clear that cells viability start to significantly decline after adding doxycycline to a final concentration of 1.2 ng/ml.

Different Copy number Measurements.

The dcas9 cassette is located on a standard pSB3K3 vector (p15A ori) and it’s expression, driven by tet repressible promoter, is remained constant upon the addition of two different concentrations of doxycycline (0.8 ng/ml, 1.2 ng/ml). We inserted CRISPRi stabilized J23104 promoter (BBa_K2839016) into plasmids with different Origins of Replication (psc101, pUC19-derived pMB1). In order to investigate their stabilization effect on sfGFP expression across different copy numbers we measured fluorescence, using sfGFP as a marker. For the sfGFP fluorescence intensity measurements, we conducted flow cytometry analysis.

Figure 4. CRISPRi stabilized promoter and non stabilized constitutive pT7A1w1 promoter flow cytometry fluorescence measurements at two different copy numbers. Error bars represent standard deviation from three biological replicates.

Stabilization of the BBa_J23104 Anderson promoter was successful, as seen in Figures 5 and 6.

Diagram image Figure 5. Time assays of CRISPRi stabilized promoter at different dCas9 induction levels and copy numbers. Flow cytometry measurements were performed at 1.5, 3.5 and 7.5 hours post-induction with doxycycline.

From figure 4 it is clear that sfGFP expression level, under the control of CRISPRi stabilized J23104 promoter (BBa_K2839016) with dCas9 levels induced by 0.8 or 1.2 ng/ml doxycycline, remains stable when expressed from different vectors, whereas, without dCas9 expression, sfGFP expression level changes when different copy number plasmids are used for its expression.

Higher induction level of dCas9 results in lower stabilization Error and lower expression. This shows that dCas9 expression partially determines the repression strength, even at seemingly not saturated levels of expression. Stabilization Error (E) for 0.8ng/ml Doxycycline induction was calculated to be approximately 0.275 and for 1.2ng/ml, approximately 0.106. Stabilization Error was calculated as the relative change in GOI expression as the copy number increases from a minimum copy number (Cmin) to infinity (approximately equal to the expression level at the copy number of pUC19-derived pMB1. This may point towards the ability of CRISPR stabilized promoters to be tuned to the desired stabilization error-relative strength level by changing the expression level of dCas9. Lower expression levels at higher dCas9 expression levels may be explained as a result of growth rate reduction and cell morphology changes from dCas9 overexpression.

Riboswitches

Aiming to incorporate translational control to our promoters we introduced two Theophylline responsive Riboswitches which we thoroughly characterized. As a reporter, we chose sfGFP fused with the first 99 nucleotides of luciferase to avoid possible changes in the secondary structure of the aptamer caused by sfGFP. The riboswitches’ characterization devices, BBa_K2839002, BBa_K2839006, were inserted into pSB1C3 vector. For the sfGFP fluorescence intensity measurements we used flow cytometry.

Figure 6. Theo27 response to differential induction by theophylline. Fluorescence was measured 7 hours after initial induction with theophylline.

The results prove that both riboswitches reacted to theophylline, facilitating inducible expression of sfGFP. Specifically, sfGFP activity increased in direct proportion to the concentration of the ligand.

Figure 7. Behaviour of 12.1 Theophylline riboswitch driving the expression of the same marker with different N-terminus amino-acid sequences.

12.1 Riboswitch does not respond to theophylline when driving sfGFP expression, but retains functionality upon fusion of the sfGFP with the first 99 nucleotides of the luciferase protein, after the start codon.

FIgure 8. 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).

The proper function of the 12.1 riboswitch under TALE promoter stabilization proves that the two systems, are, as expected (link design), orthogonal to each other. The drop in expression at 8mM theophylline, can be credited to the negative effects on growth that high theophylline concentrations (>5mM) exhibit.

From figures 7 and 8, it is evident that the 12.1 theophylline riboswitch driving sfGFP fusion protein, displays better activation ratio than Theo27 theophylline responsive riboswitch. Therefore, we incorporated it downstream of the TALEsp1-Pupsp1 stabilized promoter, achieving on the fly inducibility.

Conclusions

  • We characterized two TALE stabilized promoter variants, and proved their expected function.
  • We designed and successfully implemented a CRISPRi stabilized promoter and confirmed that CRISPRi has the potential to stabilize promoters over a wide range of plasmid copy numbers for different dCas9 expression levels.
  • Stabilized promoters do not interfere with the function of downstream riboswitches.
  • Successfully controlled stabilized gene expression with theophylline responsive aptamer.
  • Restoration of functionality of a non functional part when driving a common reporter (sfGFP), by fusing it with the 99 nucleotides of a different one.
  • We characterized 3 riboswitches.

What we unfortunately did not achieve

  • Even though we successfully cloned a heterologous AND gate and its stabilized variant into different plasmid backbones, we didn’t manage to properly characterize their functionality and behaviour over a range of different copy numbers.
  • We didn’t succeed in plotting a CRISPRisp response function to determine the cooperativity of repression and gain further insight into the behaviour of this system.
  • We did not manage to characterize a RNA-based iFFL stabilized promoter utilizing a transcriptional attenuator, due to time constraints.

Future Experiments

  • Properly stabilize and prove the improvement of the heterologous AND-gate.
  • Better characterize CRISPRi stabilized promoters over more copy numbers and in different E. coli strains.
  • Achieve promoter stabilization via the use of a transcriptional attenuator.