Team:Calgary/Secretion

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Secretion

Overview

After E. coli BL21(DE3) engineered by the Synthesis team produces poly(3-hydroxybutyrate) (PHB), it is stored as intracellular granules that range in size from 60-80 nm. This creates a challenging problem: efficient extraction of the desired PHB from inside the cell. During the early stages of our project, we explored many possible PHB extraction methods. Before deciding on using a PHB-secretion system, we evaluated the advantages and disadvantages of each system in terms of our space application:

Secretion

Secretion of PHB via the Type I hemolysin secretion pathway, which is endogenous to E. coli (Rahman et al., 2013)

Advantages:

• No chemicals are required
• PHB can be removed from media without killing cells
• Secretion is automatic
• Easier separation of PHB from media because there are minimal amounts of cellular debris
• Continuous production process

Disadvantages:

• Lower recorded yield than other methods
• Less characterized in literature than lysis mechanisms

Chemical Lysis

Traditionally uses solvent extraction employing chemicals such as chloroform and sodium hypochlorite (Hahn et al., 1994).

Advantages:

• Efficient yields
• Simple

Disadvantages:

• High cost of the transport of chemicals to and from Mars
• Requires manual input of chemicals, using precious time in an astronaut's schedule
• Lysis results in dead cells, and our system would need to be reinoculated consistently
• Cellular debris from lysis makes separation of PHB more difficult

Heat-Induced Lysis

T4 lysis genes (holins and endolysins) under the control of the pR Lambda promoter with thermosensitive repressor proteins cI587. When heated, lysis genes become activated (George et al., 1987).

Advantages:

• No chemicals required
• Lysis is automatic

Disadvantages:

• Heating our fermenters on Mars would use large amounts of precious power
• Lysis results in dead cells that can no longer replicate and produce PHB, so our system would need to be reinoculated consistently
• Cellular debris from lysis makes separation of PHB more difficult

We ultimately chose a secretion based system because of its advantages in space. This system requires no chemical input, no replacement of bacteria, and provides continuous production that saves both time and room aboard the spacecraft, which are both precious commodities during space travel.

We decided to implement a PHB secretion system that takes advantage of the naturally-occurring Type I hemolysin system found in E. coli. Intracellular PHB granules are tagged with the hemolysin secretion motif, known as HlyA, and the granule is then secreted from the cell via hemolysin membrane transport proteins. This system is advantageous for our space application because it reduces extra materials and power needed, allows cells to remain intact for easier PHB separation from the liquid media, and only requires minimal bacteria replacement. The hemolysin secretion mechanism has been used previously by the SDU-Denmark 2016 and Utah 2009 iGEM teams for PHB secretion. We have used BBa_K2018024 from the SDU-Denmark 2016 as the basis for the design of our secretion complex.

Our Secretion System

Type I Secretion

PHB is secreted through the hemolysin pathway, a Type I secretion system, that is endogenous to E. coli. Naturally, the hemolysin toxin (HlyA) is secreted from E. coli as a defence mechanism. This is a single-step process that involves three additional proteins: HlyB, HlyD, and TolC. HlyB is an active transport protein that uses ATP in the cytoplasmic membrane, HlyD is a membrane fusion protein that spans the periplasm and connects the inner and outer membrane proteins, and TolC rests in the outer membrane. When HlyB recognizes the C-terminus of HlyA, it stimulates formation of the secretion channel and the toxin is secreted (Thomas et al., 2014).

To use this system in our biologically engineered cells, we added the C-terminus of HlyA to the end of a phasin molecule. Phasin, coded for by phaP, is a small structural protein found in bacteria that naturally produce PHB. It binds to intracellular PHB, and therefore the PHB granules with bound phasin-HlyA tag are secreted as one.

Figure 1: The hemolysin Type I secretion pathway used for PHB secretion. When the HlyA secretion tag fused to phasin is recognized by HlyB, three membrane transport proteins (HlyB, HlyD, and TolC) assemble at the plasma membrane. PHB granules with the attached phasin-HlyA fusion protein are secreted as a whole from the cell.

Our Construct-Part: BBa_K2260002

To design our Biobrick for PHB secretion, we used BBa_K2018024 from the SDU-Denmark 2016 team. This part contains a coding region for phasin (phaP), originally from Ralstonia eutropha, as well as a coding region for the HlyA secretion tag, originally from E. coli. Since our chassis is E. coli BL21(DE3), we codon optimized the entire sequence for E. coli to improve protein expression and removed restriction sites to make it compatible with all iGEM RFC assembly standards. We chose to use an IPTG-inducible T7 bacteriophage promoter to upregulate the production of Phasin-HlyA Tag because it has been observed that increased production of Phasin may decrease PHB granule size, which could increase the efficiency of PHB translocation across the cellular membrane (Maehara et al., 1999). Also, genes under the control of an inducible promoter have much higher rate of transcription than those that are constitutive. The E. coli BL21(DE3) genome contains a coding region for T7 RNA polymerase under the control of an IPTG-inducible promoter, and it was therefore chosen as an ideal chassis. Furthermore, a FLAG tag (amino acid sequence DYKDDDDK) was added to the N-terminus of the Phasin-HlyA fusion protein for easier isolation of our protein during protein expression analyses.

Our BioBrick (Part: BBa_K2260002) was designed with the standard prefix and suffix so that it could be used in the pSB1C3 vector, which has chloramphenicol resistance and is the standard vector used for iGEM.

Figure 2: Genetic construct of Phasin-HlyA Tag ( Part: BBa_K2260002). BBa_B0034 is a common E. coli ribosome binding site. A FLAG tag (not visible) was also added to the N-terminus of the phasin-HlyA fusion protein (BBa_K2018024).

Experiments and Results

To quantify phasin-HlyA, we carried out two experiments: a secretion assay and SDS-Page:

Secretion assay

For this experiment, the production and secretion of PHB from E. coli BL21(DE3) transformed with either pSB1C3-phaCAB-phasin-HlyA Tag or pSB1C3-phaCAB (as a negative control) was measured. Triplicates in LB media + 3% glucose + chloramphenicol were incubated at 37°C and 150 rpm in an aerobic environment for 48 hours, then separated into secreted and intracellular fractions by differential centrifugation at 24-hour intervals. The "secreted" fraction contained the media that the cells were incubated in, and any PHB found in this fraction was therefore either secreted or released by lysed (dead) cells. The "intracellular" fraction contained all of the live cells. Before separation, CaCl₂ was added to promote aggregation and pelleting of the secreted PHB. Our method for fractionation can be found on our Experiments page and has been adapted from research conducted by Rahman et. al (2013).

After fractionation, the secreted fractions were treated with 1 % Triton X-100 in PBS to remove cellular debris that may have been pelleted with the secreted PHB, and the intracellular PHB portions were treated with sodium hypochlorite to extract PHB granules. Both secreted and intracellular PHB pellets were dried overnight and relative yield between the two fractions was compared.

After correcting for the mass of secreted PHB to account for the CaCl₂ that was added, the results show that at 48 hours there was a 114% increase in the amount of PHB secreted by cells that contain pSB1C3-phaCAB-Phasin-HlyA Tag compared to cells that contain pSB1C3-phaCAB only. However, there is very little difference between the two groups at 24 hours (Figure 3). Therefore it can be inferred that PHB secretion with Phasin-HlyA increased only after at least 24 hours of incubation and that Phasin-HlyA Tag was successful in increasing secretion of PHB by E. coli. The production of "secreted" PHB in control cells may not be PHB that was actually secreted, but instead it may be PHB that was released into the media as a result of cell death and lysis, which is a normal part of the E. coli life-cycle. More details on our experimental results can be found on our Results page!

Figure 3: Experimental results of secretion assay. (-) is used to denote negative control, pSB1C3-PhaCAB and (pSB1C3-Phasin) is used to denote PHB-secreting strains, pSB1C3-PhaCAB-Phasin-HlyA Tag. There is a 40 % increase in the amount of PHB secreted by (pSB1C3-Phasin) compared to (-) after 48 hours.

SDS-PAGE to identify production of Phasin-HlyA

To confirm protein expression we carried out SDS-Page of E. coli BL21(DE3) transformed with pSB1C3-Phasin-HlyA Tag. BL21(DE3) transformed with an empty pSB1C3 vector was used as a control. Both E. coli were incubated to an OD₅₅₀ between 0.4-0.8 then induced with IPTG. IPTG is necessary to induce the expression of T7 RNA polymerase in BL21(DE3) so that our Phasin-HlyA-Tag could be transcribed by the T7 polymerase. The culture was then separated into soluble and insoluble fractions, as well as supernatant, which we hoped would contain the Phasin protein. Initial SDS-Page experiments (Figure 4) showed too many protein bands to be able to see a band for Phasin-HlyA Tag (27 kDa), therefore we prepared anti-FLAG resin and incubated the sample fractions with it in to separate our Phasin protein from the other proteins that were present. After incubation with the resin, the sample fraction was run on an SDS-PAGE gel, the results of which can be seen in Figure 4. All lanes of the gel appeared empty, even samples from our pSB1C3-Phasin-HlyA Tag, which has an expected band size around 27 kDa.

Figure 4: Photograph of our SDS-PAGE gel electrophoresis gel run with proteins from E.coli BL21(DE3) transformed with pSB1C3-Phasin-HlyA tag (Phasin), E.coli BL21(DE3) transformed with an empty pSB1C3 vector (pSB), and a protein ladder. There were too many protein bands present to distinctly see if Phasin-HlyA Tag was present at 27 kDa.

Figure 5: Photographs of our SDS-PAGE gel electrophoresis gels run with proteins from E.coli BL21(DE3) transformed with pSB1C3-Phasin-HlyA tag (Phasin), E.coli BL21(DE3) transformed with an empty pSB1C3 vector (pSB), and a protein ladder and had been incubated with anti-FLAG resin. All lanes are empty, indicating that the anti-FLAG resin incubation had failed.

Our final attempt at SDS-Page gel will use Immunoblotting. Samples from both E. coli variants will be prepared as described above, however, the sample fractions will be incubated with an anti-FLAG rabbit antibody instead. Then, goat anti-rabbit with IgG-HRP will be used as a secondary antibody to detect the presence of FLAG-tagged proteins by a color change. HRP changes color to blue if the secondary antibody binds to the anti-FLAG rabbit antibody bound to a FLAG-tagged protein (our Phasin-HlyA Tag).

Future Directions

In the future, our team would like to test the effects of other alterations on PHB secretion, such as:

• Modify temperature, glucose % content, and incubation time to find the optimal conditions for PHB secretion with Phasin-HlyA Tag
• HlyB, HlyD, and TolC upregulation,
• Mutated versions of HlyB, HlyD, or TolC that can have increased rates of secretion, and
• The use of another secretion system (e.g. LapA) that can export larger molecules than the HlyA system.

Works Cited

George, H.J., Watson, R.J., Harbrecht, D.F. & DeLorbe, W.J. (1987). A bacteriophage lambda cI857 cassette controls lambda pL expression vectors at physiologic temperatures. Nature Biotechnology. 5: 600-603

Hahn, K.S., Chang, Y.K, Kim, B.S. & Chang, H.N. (1994). Optimization of microbial poly(3-hydroxybutyrate) recovery using dispersions of sodium hypochlorite solution and chloroform. Biotechnology and Bioengineering. 44: 256-261

Maehara, A., Ueda, S., Nakano, H. & Yamane, T. (1999). Analyses of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of paracoccus denitrificans. Journal of Bacteriology. 181: 2914-2921

Rahman, A., Linton, E., Hatch, A.D., Sims, R.C. & Miller, C.D. (2013). Secretion of polyhydroxybutyrate in Escherichia coli using a synthetic biological engineering approach. Journal of Biological Engineering. 7(24):1-9

Thomas, S., Holland, I.B. & Schmitt, L. (2014). The type I secretion pathway: the hemolysin system and beyond. Biochimica et Biophysica Acta. 18(43): 1629-1641