Orthogonality results

In Synthetic Biology, two constructions can be considered orthogonal when they only interact at specific and predictable interphases, and do not disturb each other. We have studied this desirable feature of Biobrick parts by combining two constructions in the same cell and comparing this output with the one produced by simple transformations. To do this, we used both standard fluorimetry assays and flow cytometry, which allowed to get information at the cell level. Then, we wanted to go one step further: what is the effect of a simple transformation (a plasmid with a Biobrick part) into the cell architecture? To get some insights about this, we performed a proteomic analysis in which the whole proteome of an E. coli strain transformed with a Biobrick part was compared to that of the non-transformed, control strain. Last, but not least, a detailed set of equations modeling the behavior of cells carrying two Biobrick parts was developed.

1. Standard Fluorimetry assays

We performed experiments with two different combinations of Biobrick parts containing a fluorescent protein under the control of a promoter sequence from Anderson’s promoter collection: Bb1 (GFP with strong promoter J23104) + Bb2 (RFP with strong promoter J23104) and Bb2 + Bb3 (GFP with less strong promoter J23110). In all cases, several controls were used: wild type (non-transformed) cells, simple transformations with either the biobrick or an empty plasmid, and co-transformations of the biobricks and the empty plasmids. It has to be noted that there was a high background emission of red fluorescence by the cells in all cases. Our results are shown in Figures 1 and 2.

General protocol(1)

• Strike the desired strains on LB plates with the appropriate antibiotic. Allow to grow by incubating at 37°C at least 24 hours.
• Gather a large amount of bacteria with an inoculation loop and transfer to 1 mL of LB. Incubate during 20 minutes at room temperature.
• Inoculate 100 µL of bacterial suspension in a tube containing 3 mL of LB with antibiotic. Perform three biological replicas of each combination of Biobrick parts.
• Incubate until OD₆₀₀ is between 0.1-0.3.
• Measure OD₆₀₀.
• Measure GFP fluorescence (exc.= 493nm; emis.=505nm) and RFP fluorescence (exc.= 576 nm; emis.= 592 nm) in a FP6200 spectrofluorimeter (Jasco, Easton, MD) fluorometer using standard plastic cuvettes.
• With excel, normalize fluorescence by cell density, calculate the average and standard deviation taking into account the three biological replicas, and represent the data.

Results (111)

When in co-transformation, Bb1 and Bb2 showed a clearly asymmetric expression . Bb1 was highly expressed in comparison to Bb2 (2-fold in strain XL1-Blue and 3-fold in DH5α). A similar phenomenon was observed when either Bb1 or Bb2 were cotransformed with empty plasmids. Again, less fluorescence was detected in comparison to the simple transformants. Surprisingly, this fluorescence decay was strikingly different between both E. coli strains: Bb1 strongly lowered its expression in DH5α, whereas Bb2 showed a higher decay in XL1-Blue cells.

However, the result was completely different when Bb2 and Bb3 were present in the same cell. In the XL1-Blue strain, the fluorescence intensity of Bb2 was as high as that of the single transformant, whereas the fluorescence of Bb3 –containing a less strong promoter- dropped dramatically. Fluorescence intensity of Bb3 was not detectable in strain DH5α under our experimental conditions.

With these results with co-transformants, we have to conclude that Biobrick parts, even if controlled by the same promoter and cloned into the same plasmid, are not expressed at similar rates. But, is this lack of orthogonality stable and reproducible? In other words, is the asymmetric output always the same or does it fluctuate in time or among cells? We were able to answer this question by using flow cytometry. Check our results below!

2. Flow cytometry

To study cell-level fluorescence we used flow cytometry. To prepare the samples, XL1-Blue cultures (wild type, simple transformants for Bb1 and Bb2, and cotransformants with Bb1+Bb2) were grown in LB with the appropriate antibiotic, harvested and resuspended in 1mL PBS. From now on, we will refer to Bb1 as GFP, and Bb2 as RFP.

Orthogonality at the cell level

Our first result, shown in Figure 3, is the confirmation of non-orthogonal behavior as deduced by a higher expression of GFP in comparison to RFP in cotransformed XL1-Blue cultures. There is four to five times more expression of GFP in comparison to RFP (Fig 3D). These results are similar to those we previously obtained by fluorimetry (with younger cultures, 5 h), in which an imbalanced GFP:RFP rate of about 2:1 was detected. It has to be stressed that, unexpectedly, only a fraction (around 10%) of the cells exhibited activity of both reporter proteins, indicating that the fluorescence of a culture as it is usually measured (fluorimetry) represents in fact and average of a very diverse pool of individual outputs, including mono-active cells. We plan to repeat the assay using the reverse conditions: GFP in a Kan resistant plasmid, and RFP in an Amp resistant plasmid.

Orthogonality changes in time

Figure 4 shows a clear correlation of fluorescense and time in mono-transformants. Fluorescence is very low for all samples at 5h (approximately 30% of cells transformed with Bb1 express GFP but only 1% of cells transformed with Bb2 express RFP); moderate at 10h for GFP mono-transformants (41% and 1.5% respectively); and maximum after 20h, when GFP is expressed by 80% of the cells, and RFP is expressed by approximately 45% of the cells. Regarding co-transformants, fluorescence also increased with time but even after 20h only a fraction (10%) of doubly fluorescent cells were detected.

These results indicate that higher fluorescence levels are reached when cells are reaching stationary phase and that even long incubation times fail to yield a majoritary population of co-transformed cells with dual GFP and RFP activity.

Fluorescense rates within cultures: a very stable imbalance

Our last assay aimed at answering this paradoxical question: is the lack of orthogonality a stable trait? In other words, is the GFP:RFP imbalance always the same? The answer, according to Figure 5 is clear: Yes, independent cultures exhibit a striking similarity in their flow cytometry plots. Almost half of the cell population only displays GFP activity, whereas only a relatively small fraction is either displaying RFP fluorescence or both.

3. Proteomics

Our experiments involving E. coli cells co-expressing two different Biobrick parts revealed a lack of orthogonality between them. But what is going on with all the other parts naturally present in the chassis? Does the expression of a Biobrick part interact or interfere with any of the genes expressed by the host? In order to test this hypothesis, we performed a proteomic analysis on the simplest scenario: we compared the proteome of the DH5α E. coli strain expressing Biobrick part 1 (consisting of a green fluorescent protein under the control of a constitutive promoter) with that of the same strain carrying the empty cloning plasmid and the non-transformed wild-type strain.

General protocol (2)

• Set up three independent 5-mL cultures of the strains named before as we explained above for the standard fluorimetry assays.
• When an OD value between 0.2 and 0.4 is reached, pellet the cells at 4ºC and then wash them with sterile ice-cooled PBS buffer.
• Resuspend the cells in 1 mL of ice-cooled sterile PBS buffer.

After this, we kept the tubes on ice and brought them to the Proteomics lab of the University of Valencia. The proteomic analysis was carried out in the SCSIE_university of Valencia Proteomics Unit , a member of ISCIII ProteoRed Proteomics Platform. Once there, proteins were isolated and quantified with the supervision of the technicians. Finally, proteins were digested and labeled by using the iTRAQ technology, which allowed us to work with all the samples in the mass spectrometer at a time and then assign each identification to a particular sample.

Results proteomics

A general view on the protein profile of each sample was firstly observed on an SDS-PAGE gel (Figure 6). Strong differences were detected, as expected, in the bands corresponding to the GFP and the β-lactamase encoded by the ampicillin-resistance gene of the pUC57 plasmid used for cloning.

An average of 1500 proteins per sample were identified with the mass spectrometer. Among them, 472 proteins could be properly quantified. Figure 7 shows the variation of a subset of proteins among samples. Notice that chaperone proteins are specially over-represented in the strain expressing Bb1, whereas proteins involved in response to oxidative stress are particularly abundant in the strains carrying the empty plasmid.

But this apparent variation in the proteome changes when applying some statistics. A relative comparison of the average expression level of each one of the quantified proteins in the three groups of samples (WT strain, Bb1-expressing strain, and empty plasmid-carrying strain) is shown in Figure 8. Those proteins displaying the highest alterations in their expression levels are marked with numbers and identified in the table on the right.

As deduced in Figure 7, a very low number of proteins strongly changed their expression in the strains carrying Bb1 or the empty plasmid. Several statistical tests (FDR-corrected ANOVA, T-test, p-value-weighted analysis) were performed with these proteins, and only three of them proved to be significant in all the tests. These proteins were the GFP (only present in Bb1 strain), the β-lactamase (present in both Bb1 and ᴓ strains), and the chaperone protein DnaJ, which was particularly abundant in the strain expressing Bb1. The high expression of chaperone proteins was expected, since many molecules of GFP need to be properly folded in order to avoid the formation of inclusion bodies and cellular toxicity. From this experiment, we have to conclude that the expression of a simple Biobrick part such as a fluorescent protein in an E. coli cell does not alter the proteomic architecture of the chassis in a significant way. Therefore, Biobrick part 1 could be considered orthogonal with respect to chassis' parts.

4. Conclusions

• Surprisingly, standard fluorimetry assays show an assymetric output of red and green fluorescent proteins in co-transformed cells.
• Flow cytometry confirms this asymmetric activity and strongly suggests the co-existence of sub-populations of cells with different outputs. This asymmetry proved very reproducible among cultures, and matched the predictions of our theoretical model.
• The activity of fluorescent proteins dramatically changes over time, reaching a peak when cells are in the stationary phase. Despite being under the control of the same promoter, GFP is expressed earlier than RFP. To the question “are two Biobricks orthogonal?” we thus suggest to add this other question: “when are they orthogonal?”
• Proteomic analysis reveals the robustness of E. coli confronted to heterologous gene expression: only minor proteomic changes were found compared to the controls. An orthogonal relation between Biobrick part 1 and the proteomic architecture (chassis) is thus confirmed.