Team:TUDelft/Results

Sci-Phi 29

Overview


The aim of this project is to develop Sci-Phi 29: a versatile platform to expand the repertoire of bacterial species currently used in synthetic biology. Sci-Phi 29 enables host independent replication of genetic vectors using the concept of orthogonality and ensures controllable expression of a gene of interest across different bacterial species with the use of an incoherent feed-foward loop. To achieve this goal, we investigated the bacteriophage Φ29 replication system (orthogonality module) and engineered an incoherent feed-forward loop (controllability module).

Parts Construction

Within this project we successfully constructed a library of parts that make up the Sci-Phi 29 platform, all of which have been confirmed by sequencing. In total, we have generated 20 basic parts that allowed us to create 16 composite constructs for the orthogonality module and 30 composite constructs for the controllability module. All these parts have been employed in our assays which included confirmation in vitro of each single orthogonal constructs as well as the in vitro demonstration of the replication of our linear construct, toxicity assays to ensure tightly controlled expression of the orthogonal system, testing of copy number independency across different species and demonstration of transcriptional variation, modelling of translational variation independence and quantitative analysis on codon harmonization.


Part Characterization

In vitro characterization of the orthogonal replication parts


To verify that our constructs for orthogonal replication were functionally expressed, we characterized the four proteins needed for orthogonal replication, DNAP, TP, p5 and p6 in vitro. For expressing these constructs, we used PUREfrex® (GeneFrontier Corporation, Japan). The PURE (Protein Synthesis Using Recombinant Elements) system contains all the purified components found to be sufficient to sustain the reactions involved in protein synthesis. Specifically, we employed PUREfrex®2.0 for its high protein yield and its prolonged-expression lifetime in bulk. After cell-free gene expression of each construct (3 hours, 37°C) we verified for the presence of our protein of interest by SDS-PAGE and/or mass spectrometry. For fluorescent labeling, BODIPY-Lys-tRNALys (FluoroTectTM GreenLys, Promega) was added, this binds to the translation products at the lysine residues sites.Since there are a multitude of proteins present in the PUREfrex® system, for some of our constructs, especially DNAP and TP, it was difficult to properly identify the presence of our expressed protein via SDS-PAGE. Luckily, using mass spectrometry, we were able to determine whether these proteins were present To perform mass spectrometry analysis, PURE system samples were first trypsin-digested and then analyzed by liquid chromatography-coupled mass spectrometry (LC-MS). All mass spectrometry data were normalized to the presence of EF-TU, an elongation factor that can be found in the same concentration in all PURE system reactions. The mass spectrometer looks for the mass of unique peptide sequences, and their elution time. The raw data and optimized parameters and raw data for the mass spectrometry method can be found here and here.

  • DNA Polymerase ﹀

    The expression of DNAP from our construct was confirmed by mass spectrometry, as reported by figure 1. For DNAP, the mass spectrometer screened for the following peptide sequences: ENGALGFR and LVEGSPDDYTDIK, both were detected by mass spectrometry (Figure 1), so DNAP expression from our construct was successful.

    Snow
    Forest
    Figure 1: Identification by mass spectrometry of ENGALGFR peptide (left) and of LVEGSPDDYTDIK peptide (right) in the PUREfrex sample expressing DNAP.
  • Terminal Protein ﹀

    The expression of TP from our construct was confirmed by mass spectrometry, as reported by figure 2. For TP, the mass spectrometer screened for the following peptide sequences TP: IAEIER, LVDEK, ILSYLEQYR. All three peptides were detected by mass spectrometry (Figure 2), so TP expression from our construct was also successful.

    Snow
    Forest
    Mountains
    Figure 2: Identification by mass spectrometry of IAEIER peptide (left), of LVDEK peptide (middle) and of ILSYLEQYR peptide (right) in the PUREfrex sample expressing TP.
  • Single Stranded Binding Protein (p5) ﹀

    The p5 expression starting from our three DNA constructs was confirmed by mass spectrometry as reported by figure 3. For p5, the mass spectrometer screened for the following peptide sequences: IFNAQTGGGQSFK and TVAEAASDLIDLVTR. The difference in height can be attributed to the strength of the promoters, fewer peptides were measured with decreasing strength. From this data, we can conclude that p5 expression was successful.

    Moreover, the expression of p5 (DSB) was identified by an 18% SDS-PAGE gel. From the 10-μL of expression reaction, 8 μL was loaded on the SDS-PAGE, while the other 2 μL was analyzed by the mass spectrometer for further confirmation. Click here for the protocols. An SDS-PAGE was carried out for the p5 protein with three different promoter strengths (Wild-Type, medium, and weak). As a negative control, one expression reaction was run in the absence of DNA. In figure 4, the three samples with different promoter strengths and the control can be seen on the protein gel. The presence of a band (depicted with a red star) at the expected molecular weight (13,3 kDa) confirmed the presence of the expressed p5. In the control line with no gene added, a smear background of GreenLys is visible that is distinct from gene-specific bands. The other upper band could be due to contamination in the expression reaction as it is also present in the negative control.


    Snow
    Forest
    Figure 3: Identification by mass spectrometry of FNAQTGGGQSFK peptide (left) and of TVAEAASDLIDLVTR peptide (middle) in the p5 expressed sample.
    Mountains
    Figure 4: SDS-PAGE gels of DSB (p5) after cell-free expression. Translation products were analysed by fluorescence imaging at 488 nm of an 18% gel. The bands depicted with an upper red asterisk correspond to the protein of interest with expected molecular weight. In the negative control lane where no DNA was added (most right) no specific band can be observed.
  • Double Strand Binding protein (p6) ﹀

    As for p5, the expression of p6 (SSB) was confirmed by mass spectrometry, as reported by figure 5. For p6, the mass spectrometer screened for the following peptide sequences: GEPVQVVSVEPNTEVYELPVEK and FLEVATVR. The difference in height can be attributed to the strength of the promoters, fewer peptides were measured with decreasing promoter strength.

    Furthermore the expression was confirmed by an 18% SDS-PAGE gel and mass spectrometry. Click here for the protocols. An SDS-PAGE was carried out for the three p6 encoding construct with the three different promoter strengths (Wild-Type, medium, and weak). As a negative control, one expression reaction was run in the absence of DNA. In figure 6, the three samples and the control can be seen on the protein gel. The presence of a band (depicted with a red star) at the expected molecular weight (12 kDa) confirmed the presence of the expressed p5. In the control line with no gene added, a smear background of GreenLys is visible that is distinct from gene-specific bands. The other upper band could be due to contamination in the expression reaction as it is also present in the negative control. This data confirms that we were able to express p6 successfully.

    Figure 5: Identification by mass spectrometry of FLEVATVR peptide (left) and of GEPVQVVSVEPNTEVYELPVEK peptide (right) in the p6 expressed sample.
    Mountains
    Figure 6: SDS-PAGE gels of p6 (SSB) after cell-free expression. Translation products were analysed by fluorescence imaging at 488 nm of an 18% gel. The bands depicted with an upper red asterisk correspond to the protein of interest with the expected molecular weight. In the control lane where no DNA was added (most right) no specific band was observed.

Conclusion

We were able to successfully identify DNAP, TP, p5 and p6 after in vitro expression of our constructs. The results indicate that our constructs are correct and can lead to the expression of the protein of interest. Furthermore, we were able to observe a difference in expression level when using different promoter strengths, for example, a lower amount of protein was produced when using the weak promoter.


Orthogonal Replication

For this module we also wanted to demonstrate in vitro replication of our linear construct (OriL-GFP-Kan-OriR, link to page), to show that our construct can be propagated by the phi29 replication machinery.

For this experiment, we used an in-vitro transcription-translation system (PUREfrex®2.0) supplemented with dNTPs and purified auxiliary proteins: single-stranded binding protein (SSB) and double-stranded binding protein (SBD), needed for an efficient DNA replication. We used 1 nM as the starting DNA concentration of our OriL-GFP-Kan-OriR with a twofold excess of plasmid DNA encoding for both DNAP and TP. After a 4 hour incubation at 30°C, we purified the DNA from the reaction as described in the complete protocol. The size of our construct was determined by DNA gel electrophoresis. As a control, we used samples with and without dNTPs. On our gel, we observed that our linear construct (~2,3 kb) was successfully amplified, as shown in figure 7. These results show that the phi29 replication machinery can be used for orthogonal DNA replication in-vitro. We demonstrated that our origin-flanked linear construct (OriL-GFP-Kan-OriR) could be replicated in vitro by the phi29 minimal replication machinery.



Figure 7: Purified OriL-GFP-Kan-OriR from a coupled DNA transcription-translation and replication reaction with (+) and without (-) dNTPs, amplified in vitro using the PUREfrex 2.0 system An intense band in the expected size (~2.3 kb) is observed from the +dNTPs reaction, demonstrating the OriL-GFP-Kan-OriR in-vitro replication.

Conclusion

Successful replication of our linear construct has been shown on the agarose gel (figure 7). A very intense band is visible at a size of 2.3 kb, corresponding to the size of the OriL-GFP-Kan-OriR construct. This indicates that there has been successful in vitro amplification of our construct.

Toxicity Assay


When trying to express the phi29 replication machinery in E. coli we came into contact with, Dr. Chang Liu and Dr. Julian Willis, who informed us that the expression of these four proteins needs to be tightly controlled to avoid toxic events. To find conditions where DNAP, TP, p5, and p6 are expressed in cells that can show exponential growth, we conducted a toxicity assay. We individually expressed in E. coli BL21 (DE3) pLysS cells all the four components of the phi29 machinery under three different T7 promoter variants with varying strengths. The medium T7 promoter has a relative strength of 0.64 compared to the Wild-Type T7 promoter. The weak T7 promoter has a relative expression of around 0.1. The used pLysS strain used exhibits some leaky expression, allowing limited gene expression without IPTG induction (Rosano et al., 2014). Furthermore, to better find a window of non-toxicity, we induced these constructs with different IPTG concentrations. E. coli BL21(DE3) pLysS cells have their T7 RNA polymerase (RNAP) under the control of a lac promoter. IPTG binds to the lac repressor allowing the expression of the genes which are under the control of the lac operator. Therefore, the higher the IPTG concentrations, the higher the expression of T7 RNAP and the genes under the T7 promoter.

Results

We transformed all 15 constructs from the orthogonal module separately in E. coli BL21 (DE3) pLysS cells. All cells transformed with the constructs were plated on LB agar supplemented with the corresponding antibiotics; CmR and Amp (write names). Single colonies for all constructs were grown overnight in LB with the corresponding antibiotics, and diluted the next day to have the same Optical Density (OD) at 600nm. The diluted cells were directly pipetted into a 96 well plate with different IPTG concentrations. The OD of the cells was measured over 20 hours in a plate reader. From the raw data the OD over time was plotted. The maximum slope of the exponential phase was calculated from the collected data. This maximum was normalized against the maximum slope of a strain expressing GFP with the corresponding T7 promoter. These results are plotted in bar graphs, as reported in figure 8.


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Figure 8: The graphs reported in this figure show the maximum growth rate of cells containing the 5 different constructs (DNAP, TP, p5, p6 and GFP as a control). Each construct has been studied in the presence of three different strength promoters, here indicated as weak, medium, and WT promoter. For all these conditions three different IPTG inductions were applied: Top left) no IPTG induction and Top right) 1 mM IPTG induction and Bottom left) 10 mM IPTG induction

The graphs reported in figure 8 show that the maximum growth rate of the cells containing the constructs for the four replication proteins decreases whenever the promoter strength increases. The growth rates of cells expressing GFP were normalized so the relative difference could be compared to each other. When comparing the construct containing cells and the GFP one, we can conclude that expressing our constructs of interest using a higher promoter strength results in a higher display of toxicity by the cells. In some conditions, such as cells containing constructs for DNAP and p5, the cells show higher maximum growth rates than the cells containing the GFP construct. This is especially observed when strains are grown in the presence of high IPTG, which increases the concentration of expressed proteins. This can be due to the fact that high levels of GFP expression can lead to some toxicity due to H2O2 formation (Ganini et al., 2017). Cells containing TP and p6 show a significantly lower slope than cells containing GFP expressed under a WT and medium strength promoter, indicating that these proteins are more toxic to cells than GFP. When higher IPTG concentrations are supplemented to the sample, there is a higher expression of genes under the T7 promoter. This explains why higher IPTG concentrations seem to show higher toxicity for cells containing TP and p6 when normalized to cells containing GFP. In contrast, cells containing DNAP and p5 show lower toxicity at higher IPTG contractions compared to cells containing GFP. As mentioned above, this is probably due to the fact that GFP produces H2O2, and this production is also induced with IPTG.

Conclusion

Cells containing DNAP, TP, p5, and p6 under the expression of a strong promoter show higher toxicity compared to cells where these proteins are less strongly expressed, when normalized to GFP. Cells containing DNAP and p5 do not show extra toxicity compared to cells containing GFP, while cells containing TP and p6 do show toxicity. Overall, our data (Figure 8), suggests that the ideal condition to express this protein in vivo is by using medium or low strength promoter in low IPTG induction, especially for TP and p5. Until now, we have studied some conditions for toxicity for every single construct. However, if we want to express all the components together, more finetuning will be required.


Control of gene expression levels with IFFL

Overview

The behavior of genetic parts and circuits in different bacterial species is unpredictable as it is influenced by host-dependent variations (Liu et al., 2018) . Interspecies variations (Adams, 2016), such as copy number of plasmids (De Gelder, Ponciano, Joyce, & Top, 2007), transcription rates of promoters (Meysman, et al., 2014), translation initiation rates of ribosome binding sites (RBS) (Omotajo, Tate, Cho, & Choudhary, 2015) and the codon usage of coding sequences (Sharp, Bailes, Grocock, Peden, & Sockett, 2005) influence the expression levels of genetic parts. We engineered a unique implementation of the IFFL incoherent feed-forward loop motif to achieve gene expression independent of these variables. Furthermore, the IFFL loop was shown to reduce relative level expression differences between E. coli and P. putida.

Portable T7 expression system

We have co-transformed our T7 promoter IFFL circuit with the UBER portable T7 RNAP expression plasmid (Kushwaha & Salis, 2015), demonstrating the IFFL's compatibility with systems such as this.

  • Results -- Cotransformation with portable T7 expression system ﹀

      To test the compatibility of our system with the portable T7 RNAP expression system, we first cloned our T7 promoter based optimized iFFL into low and medium copy number backbones (pICH82113, and pICH82094 respectively) from the MoClo toolkit. These plasmid backbones contain origins of replication compatible with ColE1. The clones were then co-transformed with the plasmid containing the portable T7 expression system into E. coli TOP10. Wildtype E. coli TOP10 does endogenously express T7 RNAP.

      Results

      TALE system
      Figure 9: Fluorescence histogram of cotransformations obtained by flow cytometry. Black is regular E. coli TOP10 cells, green is cotransformation of our T7 promoter based optimized iFFL , pink is cotransformation of an iFFL without repression.

      Figure 9 shows the fluorescence histogram obtained by flow cytometry. A higher level of fluorescence is observed when our plasmid is cotransformed with the UBER plasmids.

    Conclusion

    The compatibility of our iFFL genetic circuits with the portable T7 expression system suggest that a promising new step of our control of expression approach is to integrate a similar autonomously regulated T7 RNAP system to create a one-plasmid portable expression tool, which potentially will, with the input of our iFFL, exhibit constant gene expression levels across diverse bacterial cells.

Transcriptional variation

Behavior of promoters (transcriptional rates) significantly changes across different bacterial hosts (Yang S et al., 2017) . Hence, promoters either need to be re-characterized for each bacterial hosts or promoters specific to the host need to be identified. Using iFFL, we demonstrated gene expression independent of transcriptional rates when the transcription rate of both genes (TALE and GOI) maintain the same ratio, as predicted by modeling .

  • Results -- Copy number independence﹀

      The expression levels in a genetic circuit are strongly correlated to the plasmid copy number of the DNA template Segall-Shapiro et al., (2018) The amount of gene plasmid copy number can change when the plasmid is transferred between organisms. Therefore there is a need for expression levels independent of plasmid copy number if the same genetic circuit is used in different organisms. The steady-state solution of our model tells us that when our repressor binding is fully non-cooperative, we have complete independence of plasmid copy number.


      TALE system
      Figure 10: Steady-state GOI production for gene plasmid copy number 1 to 600 (genome integration to high plasmid copy number plasmid).

      The model without assumptions has the same expression level independent of plasmid copy number (figure 10). We therefore can transfer our circuit between organisms and expect the expression of the gene of interest to be independent of the changes in plasmid copy number of our orthogonal plasmid.

  • Results -- Independence to promoter strengths﹀

      To validate our model prediction, we designed T7 promoters based iFFL systems with varying promoter strengths. We compared our wild-type T7 promoter based iFFL system to a iFFL system based on a T7 promoter variant with 50% strength compared to the wild-type (Ryo Komura et al., 2018) (medium T7 based iFFL system). As a control, we express GFP without TALE regulation. We expect our iFFL systems to give the same expression level independent of the promoter strength.



      The output GFP fluorescence in E. coli was measured using flow cytometry during logarithmic growth phase after induction with 1mM IPTG. As a reference for background fluorescence, we usedE. coli BL21DE(3) cells without any plasmid. The most dense region (determined by eye) in the scatter plot (forward and side-scatter) was selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram was gated to discern between fluorescent and non-fluorescent cells, by gating the E. coli BL21DE(3) cells without any plasmid. The median of the background was subtracted from the median of the samples, and the resulting values were plotted (figure 4).

      Results

      TALE system

      Figure 11: Steady-state GFP fluorescence measurement of promoter variation using flow cytometry. The graph depicts T7 and medium T7 iFFL systems, expected to give the same fluorescence according to the model. As a control, GFP under control of an unrepressed T7 promoter was used.

      In figure 4, similar GFP fluorescence can be observed for the T7 and medium T7 iFFL systems while the unrepressed control system shows higher fluorescence. This suggests successful insulation of gene expression from change in promoter strengths.

      Raw data from flow cytometry experiments can be found here.

  • Results -- Independence to IPTG concentration﹀

      Transcriptional variations may also be caused due changes in RNAP concentrations in expression strains of different species. The effect of different concentrations of IPTG, and thus changes in E. coli BL21 DE3 in vivo T7 RNAP concentrations on the iFFL loop were tested. In unrepressed systems, the expression of the GOI is a function of IPTG concentrations. However, in our iFFL system, since the transcriptional rates of TALE and GFP are under the control of T7 promoters, both genes have a constant ratio of transcription rates, thus similar GOI expression is expected. As a control, we expressed GFP under the control of the T7sp1 promoter.

      The output GFP fluorescence in E. coli was measured using flow cytometry during logarithmic growth phase after induction with different concentrations of IPTG. As a reference for background fluorescence, we used E. coli BL21(DE3) cells without any plasmid. The densest region (determined by eye) in the scatter plot (forward and side-scatter) was selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram was gated to discern between cells that are "off" or "on" (expressing GFP or not), by gating wild-type E. coli BL21(DE3). The median of the background was subtracted and the samples were compared to each other.

      Results

      In figure 12, the GFP fluorescence of the unrepressed control changes with changing concentrations of IPTG while the iFFL system shows the same GFP expression across different IPTG concentrations. Thus, the iFFL system has been shown to insulate gene expression against changes in transcription rates (achieved by varying IPTG concentrations). There is a high difference in levels of expression between the controlled and uncontrolled samples. The difference could be due to a combination of expression of two proteins instead of one and thus partitioning of resources, and the repression by the TALE protein.

      TALE system
      Figure 12: Steady-state GFP fluorescence measurement of IPTG titration using flow cytometry. The graph depicts a T7 iFFL system, induced using different levels of IPTG, which, according to the model, should give the same result. As a control, GFP under control of an unrepressed T7 promoter was used.

      Conclusion

      We can infer from the data that with our orthogonal IFFL, we can maintain constant levels of expression in varying T7 RNAP concentrations, which further corroborates with the potential of developing a portable system integrated to our loop. Unfortunately, a great decrease in fluorescence was observed. This is likely due to still fairly high rates of expression of TALEsp1, as its expressed by the same RBS. Therefore, a next step would be the design of RBS sequences that show lower translation rates of TALE or the use of lower strength T7 promoter variants.

  • Results -- Tunability﹀

      The predictions made by modeling not only tell us that we can maintain the same level of gene expression but also that we can easily tune the expression levels by changing one of the promoters. By changing one of the promoters to another variant of T7, we can expect a different level of expression, while at the same time expect it to behave similarly when transferred between organisms. Through the use of T7 variants, we could achieve wide ranges of expression levels, which can be used to establish complex genetic circuits, while also expecting it to work similarly in different biological contexts.

      We tested the prediction by changing the promoter controlling the TALE protein to a variant of T7, which has been shown to be about 10% in strength compared to the wild-type (Ryo Komura et al., 2018). According to the model, the expression should increase.

      We measure GFP fluorescence using flow cytometry. As a reference for background fluorescence, we use E. coli BL21DE(3) cells without any plasmid. The most dense region (determined by eye) in the scatter plot (forward and side-scatter) is selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram is gated to discern between cells that are "off" or "on" (expressing GFP or not), by gating the E. coli BL21DE(3) cells without any plasmid.


      The median of the background is subtracted from the samples and are compared.

      Results

      Figure 13 suggests there is a higher fluorescence when a lower T7 promoter version is used to express the TALE protein in comparison to our systems with the same ratio in promoter strengths for both genes.

      TALE system
      Figure 13: Steady-state GFP fluorescence measurement of E. coli BL21DE(3) cells expressing our iFFL systems. The graph depicts a different T7 iFFL systems, one with both promoters T7, one with both medium strength and one where the promoter controlling of the TALE is weak. As a control GFP under control of an unrepressed T7 promoter was used.

      Conclusion

      The data suggests that we can increase expression of the gene of interest by decreasing the promoter strength of the TALE gene, through the use of a weaker T7 promoter variant. As orthogonal promoter varients will keep their ratio in strengths independent of biological context this would allow for both tunability and predictability.

Conclusion

Results above indicate successful implementation of the iFFL system to insulate from transcriptional variations. Transcriptional variations were achieved by using T7 promoters of different strengths and by induction at different IPTG concentrations. Furthermore, we demonstrated the tunability of the iFFL system to achieve different levels of gene expression. As predicted by our model, we achieved gene expression independent of changes in transcriptional rates by maintaining constant ratios of transcriptional rates of TALE and GFP genes.

Translational variation

Strengths of ribosome binding sites (RBSs) across organisms change due to differing anti-Shine Dalgarno sequences (Salis, Mirsky, & Voigt, 2009) therefore, RBSs need to be recharacterized across different bacterial species. The iFFL system can be used to achieve controllable gene expression despite differences in RBS strengths. In order to see the effect of translational variation on the expression levels of the gene of interest (GOI) we modeled our system for a range of translational rates for both genes (TALE and GOI).

  • Results﹀
      TALE system
      Figure 14: Steady-state GOI production while translation rates of both TALE and GOI are changed. The lines indicate the constant rate of the translation rates.

      As can be seen in figure 14, the kinetic model maintains the same level of GFP expression when the translation rates for both genes remain in a constant ratio. To keep the same ratio in translation rates across organisms we used the same ribosome binding site (RBS) for both genes. Using the same RBS ensures that translation initiation for both genes change in a similar manner (Salis et al., 2009), more on the design choices can be found here.

    Conclusion

    According to our model solution we can maintain the same level of GOI expression when both translation rates (for TALE and for the GOI) remain constant. We therefore designed our system to contain the same RBS in front of both TALE and the GOI.

Expression across different organisms

To achieve similar gene expression across different organisms, the iFFL system needs to be robust to changes in copy number, transcriptional, and translational rates. We experimentally demonstrated gene expression independent of transcriptional variations and through modeling, showed adaptation variations in copy number and translational variation of our iFFL system. Through the implementation of the iFFL loop using engineered broad host range promoters, we successfully demonstrated similar GFP expression across E. coli and P. putida. Thereby demonstrating gene expression insulated from variations associated with microbial hosts.

  • Results -- Expression across organisms﹀

      Broad host range promoter (PBHR) was designed by combining the conserved -10 and -35 regions from E.coli and B.subtilis (Yang S et al., 2018) and the promoter was engineered to contain a binding site for TALE repressor (PBHRsp1). Using the PBHR and PBHRsp1, we constructed an iFFL genetic circuit driving GFP expression. The circuit was transformed in E. coli and P. putida and, output fluorescence was measured by flow cytometry during logarithmic growth phase. To correct for background fluorescence, E. coli and P. putida without plasmids were used as blanks. GFP under the control of PBHRsp1 was used as a positive (unrepressed) control. As the cell morphologies of E. coli and P. putida are different they cannot be compared directly, gating was based on the most dense regions in the scatter plot for each organism. In order to compare the GFP expression levels between each organism, the background fluorescence for each organism was subtracted by its respective blank.

      Results

      In figure 15, the median fluorescence of the gated populations is plotted. A large difference in expression levels is observed between the unrepressed controls and the broad host range promoter based iFFL systems in E. coli and P. putida. However, similar levels of expression were observed from iFFL systems in E. coli and P. putida . The difference in expression levels between the unrepressed circuit is significantly higher than the difference in expression levels between the iFFL system (578530 and 2351.2, respectively).

      TALE system
      Figure 15: Comparison of expression across E. coli and P. putida. Blue shows constitutive expression and red shows the expression when our iFFL system is implemented.

      Conclusion

      The data suggests that our system succesfully decreased the differences in expression levels between E. coli and P. putida. More tests would need to be done to truly demonstrate that our system can achieve similar expression across bacterial species. However, we strongly believe that with our wet lab results in combination with modeling predictions we have laid the foundation stabilization of genetic circuits across species barriers.

Cross species codon harmonization


When heterologous proteins are expressed in new bacterial host cells, altered protein expression levels were observed due to the variance in codon usage between the original organism and the new host cell (Angov et al, 2008). To increase the expression level of the heterologous protein in the host cell, new codon optimization tools are developed. The current limitation of codon adaptation tools is that the adaptation of the DNA coding sequence can be performed for one single organism at the time. Therefore we created the first cross-species codon harmonization tool. The tool provides the user with a single DNA coding sequence that will yield the same protein expression level in different bacterial host cells. We demonstrated functional protein expression using our own harmonized coding sequence for E. coli , B. subtilis, and V. natriegens.

  • Results -- functional protein production﹀
    • To validate whether the cross-species codon harmonization results in a functional protein, we designed a cross-species codon harmonized GFP coding sequence. The sequence was harmonized using E. coli BL21(DE3) as reference organism, and V. natriegens NBRC 15636 = ATCC 14048 = DSM 759) and B. Subtilis subsp. subtilis str. 16
      The harmonized GFP was constructed through MoClo assembling with the WT t7 promoter , universal RBS, universal RBS harmonized GFP, harmonized GFP and WT terminator The assembled plasmid is transformed into E. coli BL21(DE3).
      We measure the GFP fluorescence using the Gel doc. As reference for fluorescence we use E. coli BL21(DE3) pLysS with BBa_K2918030 plasmid.

      Furthermore, to validate the harmonized GFP even more, a flow cytometer experiment was performed putting the harmonized GFP under two different promoter strength. The two promoter strength of the med T7sp1 promoter. and T7sp1 promoter were cloned in the same backbone ( pICH47761) and were paired with the same RBS (universal RBS). The fluorescence is measured with fluorescence readout from harmonized GFP by flow cytometry and E.coli BL21 DE(3) cells were used as blank. Click here for the protocol. FCSalyzer v.0.9.18-alpha was used to analyze data from the flow cytometry experiment.

      Results

      Both the positive control and the harmonized GFP showed some level of fluorescence expression. The harmonized GFP showed fluorescing cells but the positive control was almost not visual. For better validation, a flow cytometer measurement was performed.

      GFP Harmonized
      Figure ...: qualitative data of a fluorescence measurement of pelleted cells in eppendorf tubes after IPTG induction. From left to right E. coli BL21(DE3) pLysS with no plasmid (negative control), E. coli BL21(DE3) pLysS with JuniperGFP (positive control), and E. coli BL21(DE3) pLysS with harmonized GFP. A) The fluorescing image was taken using the gel doc. As seen the cell pellets are all fluorescing. B) After removing the autofluorescence by subtracting the negative control from the image, the green fluorescence cells has been visualized . The still fluorescing cell pellets are marked with a green circle. C) A 3D plot of the fluorescing cells. The height of the graph corresponds with the intensity of the measured GFP.


      The raw data of the cytoflow meter is shown in figure…. The curves represent fluorescence values of E.coli BL21 DE (3) cells (black), clones with GFP expressed from T7sp1 (red) and clones with GFP expressed from T7sp1 (blue). cytoflowharmonizedGFP
      Figure ...: Raw fluorescence data. The curves represent fluorscenece values of E.coli BL21 DE (3) cells (black), clones with GFP expressed from T7sp1 (red) and clones with GFP expressed from T7sp1 (blue).

Conclusion

The fluorescence measurements using the geldoc did not show a clear separation between the positive and negative controls. The results were insufficient to distinguish whether the harmonized GFP sequence resulted into functional protein production.

The flow cytometry data did show more quantitative data. The clear shift between the fluorescence intensity peaks implicates active GFP protein production. Therefore, we can conclude that the harmonized GFP coding sequence encodes for functional GFP proteins.



References