The primary purpose of Alpha hemolysin as used here is to provide a gate through the membrane for small molecules to enter the cytosol.
Alpha hemolysin (aHly) is a pore-forming protein produced by Staphylococcus aureus. It was rigorously characterized in the 90’s [Papers 1]. The pore is formed from an assembly of 6 protein monomers each with a molecular weight of 33.2 kDa corresponding to 293 amino acid residues. The assembled pore has an outer diameter of 10 nm and inner diameter of 1.6-4.6 nm or 5-17 water molecules across. This size is sufficient to gate the transport of molecules with molecular weight of 3 kDa.
The pore has a height of 10 nm along which there is a hydrophobic patch spanning 3 nm. This corresponds to the thickness of the non-polar section of a typical phospholipid bilayer. For this reason, aHly has often been used in the literature as a tool for confirming the formation of bilayer (as opposed to multilayer) in synthetic cells. The pore is functional if inserted from the outside in or vice versa. Introduction from the exterior can lead to lower efficiencies due to molecular jamming [Papers 2, 3].
We characterized the following behaviors of this module:
- pT7-aHly expresses aHly in cytosols.
- pT7-aHly expresses functional aHly in synthetic cells.
- Purified aHly is a useful tool for controlling the porosity of synthetic cells.
We have defined usage of this module in both cytosol and in synthetic cells.
The aHly module may be implemented by assembling the pT7-aHly DNA construct into a standard PURE reaction, following 
Assemble PURE Reactions. Alternatively, the module can be implemented using purified protein.
DNA Parts
pT7-aHly— Nucleus v0.1.0-001 Distribution Plate well G4
Protein Components
- aHly purified protein—MedChemExpress HY-P2967, resuspended to 10 uM.
Reaction construction in cytosols
A standard PURE reaction can be constructed with pT7-aHly. The reactions are constructed following the protocol described in Assemble PURE Reactions.
Component | Reaction Volume (ul) | Target |
Master Mix | ||
Solution A | 4 | |
Solution B | 3 | |
RNase I | 1 | |
pT7-aHly | 1 | 10 fmol/uL |
ddH2O | 1 | |
Total | 10 |
Reaction construction in synthetic cells
A PURE liposome can be constructed containing a cytosol expressing pT7-aHly. The reactions are constructed following the protocol described in .
Here, we demonstrate the functional insertion of aHly into the cell membrane by using it to quench a PURE reaction.
In this case, the cytosol contains the PURE system and NEB solution A (shorthand: N) along with two modules: pT7-aHly (hereafter “a”) and pT7-eGFP (hereafter “G”). The outer solution consists of a buffered, osmolarity-matched solution (hereafter “B”) (10 mM HEPES and 790 mM glucose).
As aHly is expressed in the cytosol and inserted into the membrane of synthetic cells, the small molecules required for transcription and translation diffuse into the outer solution. As a result, production of GFP is significantly reduced and should not be easily detected. In the absence of pT7-aHly, we expect to observe the production of GFP.
In the following table, reactions are denoted [OS-T1T2-IS], where OS = outer solution, T1 = template DNA 1, T2 = template DNA 2, Cy = cytosol. The details are described using the shorthand symbols defined above.
While the table below describes 2 reactions, we prepare enough master mix for 3 reactions to account for dead volume. In practice, a 10% excess should be sufficient though slightly more tedious to prepare.
Component | Reaction Volume (ul) | Total Volume (ul) |
Master Mix | ||
NEB-Sol A | 4 | 12 |
NEB-Sol B | 3 | 9 |
RNAse Inb. | 0.5 | 1.5 |
Sucrose (2 M) | 1.5 | 4.5 |
Total | 9 | 27 |
[OS-T1T2-Cy] | B-aG-N | B-G-N |
Master Mix | 9 | 9 |
DNA-aHly | 0.5 | 0.5 |
DNA-aGFP | 0.5 | 0.0 |
nuc Free H20 | 0.0 | 0.5 |
Total | 10 | 10 |
aHly is synthesized from pT7-aHly in cytosol
aHly expression in cytosol was validated by running a protein gel.
pT7-aHly . The left pair contains 5 uL of a PURE reaction while the right pair contains 2.5 uL of the same reaction. The reference ladder is marked on the left with molecular weights denoted in kDa. The bands containing aHly are indicated with the red arrows. aHly is synthesized from pT7-aHly in synthetic cells
We characterized the production of aHly from pT7-aHly and functional insertion into the membrane by performing time lapse imaging using two-channel epifluorescence microscopy (see 
Light Microscopy of Liposomes).
We found that the expected behavior occurs. When aHly is present, GFP production is quenched within synthetic cells. However, when the pT7-aHly template is not present, GFP is produced in a subpopulation of cells. These samples were incubated at 37 C for 7.5 hours.
For synthetic cells containing PURE, it becomes important to consider the outer and inner solutions since the necessary small molecule building blocks for transcription and translation will be lost. This behavior has been described in Papers 2 and 4.
We have found that high concentrations >100 nM of alpha hemolysin protein is associated with an increased tendency to aggregate.
We are still characterizing the interplay between osmolarity differences across the boundary and aHly on the synthetic cell population.
- Definitive structural characterization of the heptameric aHly membrane pore assembly - [Science, 1996]
- First use of aHly in a synthetic cell to extend the lifetime of a transcription translation reaction - [PNAS, 2004]
- A region of aHly is modular and can be engineered to modify its properties for the benefit of synthetic cell construction [bioarXiv, 2023]
- Transcription and translation in synthetic cells depends on the composition of the outer solution [Langmuir, 2012]
