Secretion

Overview

After the E. coli BL21 (DE3) engineered by the Synthesis subgroup has produced polyhydroxybutyrate (PHB), it is stored as intracellular granules that range in size from 60-80nm. 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 is 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 used solvent extraction, using chemicals such as chloroform and sodium hypochlorite (Hahn et al., 1994).

Advantages:

• Efficient yields
• Simple

• Transport of chemicals to and from Mars is high cost
• Requires manual input of chemicals
• Lysis kills cells, (would need to ship stocks of our engineered E. coli to Mars)
• Cellular debris from lysis makes separation of PHB more difficult

Heat-Induced Lysis

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

Advantages:

• No chemicals required
• Lysis is automatic

• Heating our fermenters on Mars would use huge amounts of power
• Lysis kills cells, (would need to ship stocks of our engineered E. coli to Mars
• Cellular debris from lysis makes separation of PHB more difficult

We ultimately chose a secretion based system, due to the impact of its advantages in space; a system that requires no chemical input, no replacement of bacteria, and provides continuous production that saves both time and room aboard the spacecraft, both of which are 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, then the granule is secreted from the cell via hemolysin membrane transport proteins. This system is advantageous for our space application because it reduces extra materials/power needs, allows cells to remain intact for easier PHB separation from the liquid media, and only minimal bacteria replacement would be required. The hemolysin secretion mechanism has been used previously by the SDU-Denmark 2016 and Utah 2009 iGEM teams for PHB secretion. We have used Part: BBa_K2018024 from the SDU-Denmark 2016 as the basis for the design of our secretion complex.

Our Secretion System

Type 1 Secretion

PHB is secreted through the hemolysin pathway, a type 1 secretion system, that is endogenous to E. coli. Naturally, hemolysin toxin (HlyA) is secreted from the E. coli as a defense mechanism. This is a single step process involving three main 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, 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.

Our Construct

To design our Biobrick for PHB secretion, we used Part:BBa_K2018024 from the SDU-Denmark 2016 team. This part contains a coding region for phasin (PhaP), originally from Ralstonia eutropha, and a coding region for 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 RFC assembly standards. We chose to use a T7 phage promoter to upregulate the production of phasin-HlyA tag because it has been observed that up-regulated production of phasin may decrease PHB granule size, which could increase the efficiency of PHB translocation across the cellular membrane (Maehara et al., 1999). Genes under the control of a T7 promoter have much higher rate of transcription than those that are under the control of native E. coli promoters. BL21 (DE3) contains genes for T7 RNA Polymerase, therefore it was chosen as an ideal chassis. Furthermore, a FLAG Tag (amino acid sequence DYKDDDDK) was added to the C-terminal end of the Phasin-HlyA fusion protein for easier isolation of our protein during protein expression analyses.

Our biobrick was designed with the standard prefix and suffix so that it could be used in a pSB1C3 vector, which has chloramphenicol resistance and is the standard vector used for iGEM.

Experiments

To quantify Phasin-HlyA Tag we carried out two experiments: SDS-Page and a secretion assay.

SDS-Page to identify production of Phasin-HlyA Tag

This experiment involved the use of electrophoresis to isolate for phasin-HlyA protein from our E.coli BL21(DE3) transformed with pSB1c3-Phasin-HlyA. We used E.coli BL21(DE3) transformed with an empty pSB1c3 vector for our negative control. After incubating both E.coli variants to an OD550 of 0.4 to 0.8, we inoculated them with IPTG to induce the production of T7 polymerase, which consequently caused transcription of the phasin-HlyA insert due to our T7 promoter. The resulting culture was centrifuged to separate the supernatant from the pellet and both placed in -20°C freezer to induce lysis. The supernatant here would contain any proteins that would have been secreted by the cell. The pellet samples were then lysed using a lysozyme (L6876 SIGMA) in a STET buffer. The resulting mixture was again centrifuged to separate the supernatant and the pellet. The supernatant here would contain the soluble proteins fraction and the pellet would contain the insoluble protein fractions. All three fractions from the two E.coli variants (and their replicates) were run using the SDS-PAGE gel electrophoresis protocol outlined in the

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 48h, then separated into secreted and intracellular fractions by differential centrifugation at 24h intervals. 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 intracellular PHB portions were treated with sodium hypochlorite to extract the PHB granules. Both secreted and intracellular PHB pellets were dried overnight and relative yield between the two was compared.

Future Directions

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

• HlyB, HlyD, and TolC upregulation.
• Mutated versions of HlyB, HlyD, or TolC that can have increased rates of secretion.
• Use of another secretion system (eg: LapA) that can export larger molecules than the HlyA system.