Team:IISER-Bhopal-India/approach

Team Methnote

Our Approach - Motivation, Methodology and Expected Pay-Offs

The goal of developing MethNote was to build a cost effective and reliable detector for local levels of methane. As laid down in our ‘Idea’ section, the aim was to introduce the genes needed to convert Methane to Methanol into our chassis Pichia pastoris along with a sensor for detecting levels of methanol.

The main problem we were facing was that the enzyme which was needed to convert Methane to Methanol (sMMO- soluble Methane Monooxygenase enzyme, having six subunits, from Methylotrophic bacteria Methylococcus capsulatus) had to be cloned into our chassis organism [1]. So, a traditional cloning approach would not be viable since we needed to find six vectors (one for each subunit) carrying unique antibiotic resistance markers to clone the genes. Such a stock of vectors is simply not commercially available. Hence, we needed to reduce the number of vectors. A logical idea was to increase the number of genes being cloned into one vector and so we decided to use the SOEing PCR method [2] to join two genes together and put them into one vector carrying a unique antibiotic marker. This brought the number of vectors required down to three and we settled upon using shuttle vectors namely pGKB, pGHYB and pGAPZ B for E. Coli and P. pastoris carrying the resistance markers for Kanamycin, Hygromycin and Zeocin, all proven to inhibit the growth of P. pastoris at certain concentrations.

Figure 1: Shuttle Vectors for our cloning strategy. The empty vector backbone of pGKB (3506 bp) [left], pGHYB (3642 bp) [center] and pGAPZ B(2882 bp) [right] with Kanamycin, Hygromycin and Zeocin selectable markers respectively.

An immediate problem we faced was that these vectors were for cloning a single gene since they contained a unique promoter and terminator sequence. Our idea was to clone the terminator sequence and put it after Gene 1 and clone the promoter sequence behind Gene 2. After that, we would use SOEing PCR and complementary overhangs to amplify the product containing the two genes along with their own promoters and terminators and ligate that into our vectors.

Figure 2: Schematic for cloning startegy using SOEing PCR 6xHis-tagged Gene 1 along with AOX terminator has been cloned using one set of restriction enzymes and 6xHis-tagged Gene 2 with GAP promoter has been cloned using another set of restriction enzymes.

For the methanol reporter, we decided to clone the eRFP gene into the vector pHILD-2 carrying the AOX promoter, which would sense methanol, and also carry 6xHis marker. To select this vector after cloning, we decided to use P. pastoris strain GS115 which is a His-mutant.

Figure 3:The empty vector backbone of pHI-LD2 vector (8120 bp) with HIS4 gene resposnible for histidine biosynthesis.

After we got the SOEing products cloned into the vectors, we would co-transform them into the yeast and grow them in media containing the chosen antibiotics. These cells would be harvested and we would check for protein expression using Western Blotting Assay along with anti-6xHis antibody since all the sMMO genes are already tagged with 6xHis tag at the C-terminus.

The final step would be to clone the promoter-reporter construct into the chassis and grow them in minimal media containing the chosen antibiotic supplemented with Histidine and Sorbitol and lacking glucose since glucose is known to inhibit transcription from the AOX promoter but Sorbitol allows it [3].

These cells would be grown in chambers containing fixed amounts of Methane to air ratio and the fluorescence would be measured for each to help calibrate our device for use in the field.

An Alternate Strategy

During the course of our experiments, we came across the Viral 2A Peptide system which has the potential of reducing the number of required plasmids even further. 2A peptides are about 20 amino acids long and when they are inserted between 2 genes in a Eukaryotic system, they cause the ribosome to skip the formation of a peptide bond, thus creating 2 polypeptides from the same mRNA chain (just like in Prokaryotes)[4]. The beauty is that no separate promoters or terminators are required for each separate gene which saves a lot of space in the plasmid.

Figure 4: Overview of Viral 2A Peptide system. Each part (gene)has been linked using viral 2A peptide sequence.The genes are transcribed as a single polycistronic mRNA. The part( protein) is translated as individual proteins with 2A Peptide sequence attached to its C-terminal

We are planning to use the P2A system as an alternative to the SOEing system by ordering synthetic constructs from IDT. These constructs would contain three sMMO genes each along with P2A sites in between. The constructs would be made such that the total size is less than 3kb (since that is the maximum size permissible for a gBlock from IDT).

Figure 5: These gBlocks would be amplified and simply cloned into the desired vectors for expression in P. pastoris.

References:

1. Merkx, M., Kopp, D.A., Sazinsky, M.H., Blazyk, J.L., Müller, J. and Lippard, S.J., 2001. Dioxygen activation and methane hydroxylation by soluble methane monooxygenase: a tale of two irons and three proteins. Angewandte Chemie International Edition, 40(15), pp.2782-2807.

2. Bryksin, A.V. and Matsumura, I., 2010. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques, 48(6), pp.463-465.

Pichia pastoris

4. Liu, Z., Chen, O., Wall, J.B.J., Zheng, M., Zhou, Y., Wang, L., Vaseghi, H.R., Qian, L. and Liu, J., 2017. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Scientific reports, 7(1), p.2193.

iGEM