Part:BBa_K4367009
Inhibited Beta-galactosidase (iGal)
iGal is a designed reporter-protein in the Cell-free system that catalyzes X-gal into a blue pigment when it gets activated by Tobacco Etch Virus Protease (TEVp).
Description
Inhibited Beta-galactosidase (iGal) is a synthetic protein that was developed and based on the tetrameric protein Beta-galactosidase (B-gal) and the phenomena behind Blue-White Screening, namely Alpha-complementation [1]. B-gal is an enzyme that is expressed by E. coli through the LacZ operon, and in its natural environment B-gal cleaves lactose into glucose and galactose for the cell [2]. B-gal is also able to cleave an artificial molecule, X-gal, which when cleaved produces a blue pigment. Alpha-complementation is when two mutated and enzymatically inactive fragments of B-gal, an Omega-fragment and an Alpha-fragment, can bind to each other and form a functional monomer [3]. That monomer can in turn form an enzymatically active tetramer with the same function as B-gal. There exists multiple possible sequences of the Omega- and Alpha-fragments that can undergo Alpha-complementation [3]. The chosen sequence of the Omega-fragment is B-gal that has amino acids 11-41 deleted [3, 4] and the chosen sequence of the Alpha-fragment are the first 75 amino acids of B-gal [4]. This is visualised in figure 1.
Figure 1. The Alpha- and Omega-fragments visualised in relation to the Beta-galactosidase protein.
Our simulations with AlphaFold suggested that a free floating Alpha-fragment protein would bind itself to the Omega-fragment where the amino acids had been deleted from B-gal but were present in the Alpha-fragment. In other words, the Alpha-fragment bound itself close to the N-terminal of the Omega-fragment. Our simulations in AlphaFold also suggested that the N- and C-terminals of the Omega-fragment are located on opposite sides of the protein. With this in mind, iGal was designed by fusing the Omega- and Alpha-fragments, putting the Omega-fragment at the N-terminal and Alpha-fragment at the C-terminal, being connected by a cleavable linker, as shown in figure 2. iGal was designed such that in its natural state, the Alpha-fragment would be separated from the place on the Omega-fragment that could be complemented by the Alpha-fragment. This design was chosen to prevent spontaneous Alpha-complementation, but still being possible to induce Alpha-complementation if the cleavable linker would be cut by a protease. The cleavable linker consisted of a recognition site (ENLYFQ/G) for Tobacco Etch Virus Protease (TEVp) [5] flanked by a flexible linker (GGGSG) [6] on each side. The design of iGal was to essentially make it an inhibited version of ‘B-gal’, until it got into contact with a TEVp, which would restore its enzymatic function and make it an active ‘B-gal’.
Figure 2. The design of iGal.
The iGal also has a 6xHis-tag at the N-terminal, allowing for protein purification. The part is also adapted for Modular Cloning as it has flanking Part-3-overhangs.
Usage
Wild type B-gal is able to catalyse the breakdown of X-gal and produce a blue pigment [1] and an activated iGal should be able to do the same. The following schematic in figure 3 shows how iGal is supposed to look like and behave in the presence of TEVp and X-gal. The large red piece is the Omega-fragment, the purple piece is the Alpha-fragment, the yellow circle is TEVp. TEVp would cleave iGal and the fragments would undergo Alpha-complementation to form a monomer. The monomers would form an enzymatically active tetramer that would convert the colourless X-gal into a blue pigment.
Figure 3. Schematic of the iGal-system.
iGal along with X-gal would act as a reporter if there is catalytically active TEVp in the solution, and our Cell-free system would induce catalytic activity of TEVp if it would get into contact with target DNA.
Future design considerations
Colab-AlphaFold was used initially to examine the folding of iGal, but it was unable to simulate the whole iGal as it was too big. When the whole iGal was run in AlphaFold, potential problems came to light. There exists a risk that iGal is always active. The reason for this is that a linker is possibly too long and could allow for the Alpha- and Omega-fragments to complement each other despite the linker not being cleaved, which is shown in figure 4.
Figure 4. iGal (left) compared to B-gal [PDB:1F4H] (right).
If iGal would always be active because of this, iGal would have to be redesigned, possibly by just shortening the GS-linker. So if iGal and X-gal would always give a positive result, even without TEVp, this could be a solution. It would be a good idea to use AlphaFold to see what length of the GS-linker would not result in the Alpha-fragment reaching the spot on the Omega-fragment that would result in alpha-complementation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3319
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 14
Illegal BsaI.rc site found at 3326
Characterization
The experimental setup tested the mixtures of X-gal and cell-lysates from S. cerevisiae transformed with TEVp or iGal. Each column corresponds to one transformation of S. cerevisiae with TEVp and each row makes up one transformation with the iGal. As negative control, no TEVp and iGal were added to the last column and row respectivly.
The first experiment was performed according to the specified experimental setup and it used MilliQ containing X-gal as buffer. The result is shown in figure 5. Going from left to right, the pictures show how the experiment looks after 1, 2, 3, and 6 days since its start.
Figure 5. An experiment mixing lysed cells that were with iGal and TEVp, with X-gal and MilliQ as buffer.
The second experiment used the same experimental setup, but the MilliQ buffer was replaced with PBS. The result is shown in figure 6. Going from left to right, the pictures show how the experiment looks after 1, 2, and 5 days since its start.
Figure 6. An experiment mixing lysed cells that were with iGal and TEVp, with X-gal and PBS as buffer.
A detailed analysis of the experiment can be found in Results, but a conclusion was that the lysed cells of S. cerevisiae contained something that degraded X-gal. This made it not possible to conclude if TEVp and/or iGal were expressed or were functional.
References
[1] GoldBio Protocol (2018). Blue-White Screening of Bacterial Colonies. Utilizing X-Gal and IPTG Plates.
[2] Douglas H Juers, Brian W Matthews, and Reuben E Huber (2012). LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575911/
[3] Irving Zabin (1982). β-Galactosidase α-complementation. Available at: https://link.springer.com/article/10.1007/BF00242487
[4] iGEM07_USTC (2007). Part:BBa_I732006 - lacZ alpha fragment. Available at: https://parts.igem.org/Part:BBa_I732006
[5] David S. Waugh. TEV Protease FAQ. Available at: https://structbio.vanderbilt.edu/wetlab/private/vectors/Tev/tevnotes.Waugh.pdf
[6] Xiaoying Chen, Jennica Zaro, and Wei-Chiang Shen (2012). Fusion Protein Linkers: Property, Design and Functionality. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726540/
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