Standardization

Biobrick parts are supposed to be standard, this is, they are expected to behave in a similar and predictable way in different host organisms or “chassis”. In this part of the project we used six different E. coli strains commonly used in molecular biology labs to test whether the behavior of our Biobrick parts was homogeneous among them. Surprisingly, only one of the constructions we tested displayed a relatively standard output among the different strains.

General protocol

• 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 replica of each culture. The expression of Bb4 was induced with a heat shock at 42 °C for 2 min. The expression of Bb5 and Bb6 was induced by adding doxycycline and IPTG, respectively, to the medium.
• Incubate until OD₆₀₀ is between 0.1-0.3.
• Measure OD₆₀₀ or OD₅₉₅.
• In the case of Biobrick parts 4-6, sonicate an aliquot of the culture, eliminate cell debris, add X-Gal, incubate 6 min at RT, and stop the reaction by adding Na₂CO₃.
• Measure either GFP fluorescence (Bb1 and Bb3; exc.= 493nm; emis.=505nm), RFP fluorescence (Bb2; exc.= 576 nm; emis.= 592 nm), or absorbance at 630 nm (Bb4, Bb5, and Bb6). Fluorescence measurements were performed in a Hitachi F7000 plate reader, whereas a spectrophotometer was used for absorbance measurements.
With Excel, normalize fluorescence by cell density, and calculate the average and standard deviation taking into account the three biological replicas. Represent the data.

Orthogonality

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. According to our experiments, that were carried out in two different E. coli strains, Biobrick parts do not behave orthogonal when present in the same cell, being the expression of one of the parts 2 to 3 times higher than the other one. Then, we wanted to go un 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 are currently waiting for a proteomic analysis in which the whole proteome of an E. coli strain transformed with a Biobrick part will be compared to that of the non-transformed, control strain.

General protocol

• 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

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. In all cases there was a high background emission of red fluorescence by the cells. Our Results are shown in Figures 1 and 2.

Figure 1. Fluorescence emission in XL1-Blue E. coli strains co-expressing Biobrick parts 1, 2, and 3 reveals asymmetry of behaviors (lack of orthogonality). Left, cells containing both Biobrick parts 1 and 2, Biobrick part 1 and an empty kanamycin plasmid (e.k.p), Biobrick part 2 and an empty ampicillin plasmid (e.a.p.), Biobrick part 1, and Biobrick part 2. Right: Cells containing both Biobrick parts 2 and 3, Biobrick part 2 and an empty kanamycin plasmid (e.k.p), Biobrick part 3 and an empty ampicillin plasmid (e.a.p.), Biobrick part 2, and Biobrick part 3. XL1-Blue E. coli cells containing no plasmids or empty plasmids were used as controls. Both green (505 nm) and red (592 nm) fluorescence intensity (FI) were measured for all samples and normalized by the optical density at 600 nm (OD600).

Figure 2. Fluorescence emission by DH5-α E. coli strains co-expressing Biobrick parts 1 and 2 also reveals non orthogonal expression of Biobrick parts. Cells containing both Biobrick parts 1 and 2, Biobrick part 1 and an empty kanamycin plasmid (e.k.p), Biobrick part 2 and an empty ampicillin plasmid (e.a.p.), Biobrick part 1, and Biobrick part 2 were analyzed. XL1-Blue E. coli cells containing no plasmids or empty plasmids were used as controls. Both green (505 nm) and red (592 nm) fluorescence intensity (FI) were measured for all samples and normalized by the optical density at 600 nm (OD600).

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? In other words, is the asymmetric output always the same or it fluctuates depending on the culture? We designed a second experiment to answer this question. By performing several subcultures of the co-transformant strains -with five independent replica of each- and measuring the fluorescence output of each replicate after the subculturing steps, we will check whether fluorescence asymmetry is stable. On the other hand, we have only studied the average fluorescence in a population of cells in our experiments… but what are the variations between single cells? A simple experiment using flow cytometry can help us solve this question…