Fine-tuning PHB expression by manipulating RBS strength
Our work aimed to improve upon the University of Calgary’s phasin-HlyA part (BBa_K2260002) submitted in 2017. Phasin is an intracellular protein native to PHB-producing bacteria, which coats and stabilises polyhydroxybutyrate (PHB) granules in the bacterial cytoplasm. Hemolysin A (HlyA) is a native toxin in E. coli, and is secreted via the single-step Type I secretion system (TISS) in gram-negative bacteria (1). By adding the C-terminus of the HlyA gene to the end of the phasin molecule, the HlyA-tagged phasin was successfully secreted out of the cell via Type 1 secretion. Our aim was to improve phasin-HlyA secretion levels by modifying the strength of the ribosomal binding site (RBS) upstream of the phasin-HlyA coding sequence.
To improve the mechanical strength of bacterial cellulose (BC), the key polymer in our project, we added polyhydroxybutyrate (PHB), a polyhydroxyalkanoate and popular bioplastic. As such, we used the PHB-producing plasmid (BBa_K934001) submitted by the Tokyo Tech team in 2012, which produces intracellular PHB. However, the use of this plasmid on an industrial scale is implausible: the extraction of intracellular PHB is expensive and time consuming, and current purification methods are toxic (2).
Figure 1. PHB biosynthesis pathway in E. coli. (a) Glucose is metabolised into pyruvic acid by glycolysis. (b) Pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. (c) β-ketothiolase (coded in phaB) activity converts acetyl-CoA into acetoacetyl-CoA. (d) Acetoacetyl-Co-A is converted into R-3-hydroxybutyratyl-CoA by NADPH-dependent R-3-hydroxybutyratyl-CoA (coded in phaA). (e) R-3-hydroxybutyratyl-CoA is polymerized by PHA synthase (coded in phaC) to form PHB.
While the University of Calgary 2017 team’s phasin-HlyA part (BBa_K2260002) built on this work to secrete PHB into the extracellular space, our aim was to evaluate if we could further improve this secretion system to increase subsequent PHB production and secretion levels. Based on our team’s extensive work incorporating proteins into BC with a co-culture, and our uniaxial testing results regarding the effect of PHB on BC, the ability to increase the amount of PHB secreted and incorporated into BC would have significant implications for the industrial applications of our project.
In order to change and ideally increase PHB secretion, we turned our focus to phasin-HlyA production levels. Given the role of phasin and Hly-A in PHB granule size and secretion, we hypothesized that fine tuning the expression levels of this part would result in a higher yield of extracellular PHB.
To accomplish this, we designed four parts with varying RBS types and strengths upstream of the phasin-HlyA coding sequence. The RBS plays an important role in protein synthesis, as it binds the ribosome and positions it for accurate translation. The RBS types selected for this experiment were sourced from the iGEM Community Collection. Their relative strengths are as follows:
Table 1. The four RBS types incorporated into our construct design and their relative strengths.
Importantly, the B0034 is the same RBS used in the original phasin part (BBa_K2260002) around which we based our design, and which we aimed to improve. This will act as a positive control, against which to measure and compare the expression levels of the other parts. The additional design of a construct containing our DeadRBS (BBa_K4437500) will serve as a negative control, as the DeadRBS is non-functional RBS with low affinity for the ribosome, and should translate negligible amounts of phasin.
This concept culminated in the design of four phasin parts, each containing one of the four RBS types (Figure 2).
Figure 2. The composite part suite for the proposed phasin and PHB improvement. a) The negative control, DeadRBS-Phasin-HlyA (BBa_K4437501), containing our DeadRBS (BBa_K4437500). b) The proposed improvement, B0030-Phasin-HlyA (BBa_K4437502), with the relatively strong RBS BBa_B0034. c). The positive control, B0034-Phasin-HlyA (BBa_K4437503), with the same Bba_B0034 RBS as the original part. d) The proposed weaker expression system for comparison, B0035-Phasin-HlyA (BBa_K4437504), with the relatively weaker RBS BBa_B0035.
Overall, by testing the effect of increasing RBS strengths on phasin expression levels, and for the purposes of Cellucoat the result of overexpressing phasin was to increase PHB production and secretion for the purposes of improving the mechanical properties of the bacterial cellulose packaging material. Furthermore, by creating different constructs with varying strengths of RBS, the amount of PHB synthesised and secreted can be controlled allowing for the properties of the BC and PHB composite to be fine-tuned. The different constructs will also be useful for future teams to choose how much PHB they want to produce or secrete for their specific purpose.
A phasin-hlyA construct and primers that have the four different RBS sites (BBa_B0030, BBa_B0034, BBa_B0035, and DeadRBS or BBa_K4437500) and RE sites were created. There were two variations of the primers created with different cut sites based on the enzymes used, however it was evident that one of the cut sites between PstI and SpeI were not yielding successful digestion because the cut sites were too close to each other (~10 bp apart). Hence, the BstAPI, and BstBI RE and cut site primers were used to insert the complete phasin-hlyA construct with their respective RBS into the tokyo plasmid. The resultant constructs were: BBa_B0030-phasin-hlyA (BBa_K4437502), BBa_B0034-phasin-hlyA (BBa_K4437503), BBa_B0035-phasin-hlyA (BBa_K4437504), and DeadRBS-phasin-hlyA or BBa_K4437500-phasin-hlyA (BBa_K4437501).
A diagnostic digestion was performed, using PstI, which indicates that one band of length ~7100 bp should appear if the digestion and ligation was successful, while if the diglig was unsucessful than the bands would be a band size of 6400 bp.
Figure 3. Samples BBa_B0030-phasin-hlyA (BBa_K4437502) (2 and 5), BBa_B0034-phasin-hlyA (BBa_K4437503) (10, 11, 12), BBa_B0035-phasin-hlyA (BBa_K4437504) (16), and BBa_K4437500-phasin-hlyA (BBa_K4437501) (20) all had bands appear in the desired band size of ~7100 bp, indicating that for B0030, it was successfully digested, ligated, and transformed into TOP 10 cells, while for the other samples it was been transformed into BL21 cells. Due to time restrictions, the BBa_B0030-phasin-hlyA (BBa_K4437502) was not transformed into BL21 E.coli cells.
For the purposes of this specific subproject, the amount of the three PHB producing enzymes is irrelevant, so what was looked for in the Western blot was the presence of phasin alone with a molecular mass of 20.960 kDa, hlyA alone with a molecular mass of 6.151 kDa, or a phasin-Hlya fusion protein with a molecular mass of 27.208 kDa. These three combinations of the product of the phasin-Hlya gene are possible because Hlya is cleaved off once phasin is secreted through the Type I secretion system. Therefore, in the western it can be expected the three combinations of the phases-Hlya protein would be evident.
Figure 4. The expression of phasin-hlyA with varying RBS strengths from the PHB construct (BBa_K934001) from BL21 (DE3) E.coli strain was autoinduced for 24 hours. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. The gel was stained with Coomassie blue. The gel was loaded as follows: (1) Ladder, (2) LanM positive control (BBa_K3945001), (3) BBa_K4437500-phasin-hlyA (BBa_K4437501), (4) BBa_B0034-phasin-hlyA (BBa_K4437503), (5) BBa_B0035-phasin-hlyA (BBa_K4437504), (6) Tokyo 2012 PHB construct only (BBa_K934001).
Since there were two distinct bands at ~27 kDa and ~20 kDa in the three plasmids that contained the phasin-HlyA inserts there is phasin-hlyA being produced. However, the strength of the banding was not consistent with what was expected based on the relative strengths of the RBS placed upstream of the phasin-hlyA gene. Hence, another batch of transformed BL21 E.coli were autoinduced over 48 hours instead of 24 hours in hopes that there would be a more apparent difference in the amounts of phasin-hlyA produced between the different RBS. This is because swapping the RBS was intended for a fine-tuning effect. The cell lysate was used for an SDS to better compare the amount of phasin-hylA produced using anti-FLAG antibodies.
Figure 5. The expression of phasin-hlyA with varying RBS strengths from the PHB construct (BBa_K934001) from BL21 (DE3) E.coli strain autoinduced for 48 hours on an SDS page using Anti-FLAG antibodies and the complementary capture antibody. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. The gel was loaded as follows: (1) Ladder, (2) Tokyo 2012 PHB construct only (BBa_K934001) (3) BBa_K4437500-phasin-hlyA (BBa_K4437501), (4) BBa_B0034-phasin-hlyA (BBa_K4437503), (5) BBa_B0035-phasin-hlyA (BBa_K4437504).
The final SDS page using anti-FLAG antibodies and the complementary capture antibody yielded results that aligned with the expected amounts of phasin-hlyA produced using the varying strengths of RBS. The two bands were also consistent with the expectation that the cleaved phasin with the FLAG tag attached to the N-terminus of the protein would have a size difference of approximately 6.151 kDa compared to the phasin-hlyA fusion protein before cleavage of hlyA.
The strongest RBS used, BBa_B0034-phasin-hlyA (BBa_K4437503) had the most saturated and thickest band, indicating that phasin-hlyA translation was upregulated compared to the weaker RBS. The BBa_B0035-phasin-hlyA (BBa_K4437504), which is slightly weaker than the BBa_B0034 RBS showed slightly lower levels of phasin-hlyA synthesis, demonstrated by the lighter bands of both the cleaved and fusion phasin-hlyA protein. Lastly, the DeadRBS or BBa_K4437500-phasin-hlyA (BBa_K4437501) had levels of phasin-protein production near that of the negative Tokyo 2012 PHB (BBa_K934001) control. The slight bands in negative Tokyo 2012 PHB (BBa_K934001) control lane 2 can be attributed to leakage as its bands are similar to that of the DeadRBS-phasin lane (BBa_K4437500) which should have little to no protein synthesis.
The purpose of experimenting with different strengths of RBS upstream of the phasin-hlyA gene was to create a set of constructs that can be used to fine-tune PHB expression and secretion by up or down-regulating phasin translation. The results indicate that the stronger the RBS upstream of the phasin-hlyA gene will upregulate the amount of phasin produced. Having established that swapping out RBS even with small strength differences will have an evident impact on the amount of phasin produced, the next steps will be to determine the impact of the varying levels of phasin production on overall PHB production and secretion levels. Once the impacts of the different levels of phasin production on PHB production and secretion have been studied, the impacts of different concentrations of recombinant PHB synthesis in E.coli integrated in a coculture with K.xylinus on the mechanical properties of a PHB and BC composite will be determined using uniaxial testing.