We planned the time to be devoted for experiments and the experimental workflow based on the amount of time we predicted we would have for doing our wet-lab experiments. The experiments had been designed such that they provide an overview of our project’s proof of concept with the help of cassettes designed.
To do so, we decided to divide our wet lab work into three phases.
While dividing these phases, the first priority was given to the complexity of the cassette designed whose working has to be checked. Then it got more oriented towards the priority of the experimental results needed to move to the next parts of the wet lab workflow.
- srtf1+ mcherry: Steroid-Responsive Transcription Factor 1 linked by using Linker with mCherry Red Fluorescence Protein with the help of cloning
- ASTL+sfgfp: Astacin-like metalloendopeptidase (Human ovastacin) linked by using Linker with Superfolder Green Fluorescence Protein with the help of cloning
- P22 repressor+Azurite: P22 repressor linked by using Linker with Azurite Blue Fluorescence Protein with the help of cloning
Note- In all the above cassettes, we used a P2A peptide linker from porcine teschovirus-1 (PTV) with self-cleavage properties to make non-fusion coexpression of protein. As we wanted to detect the expression of desired proteins that are SRTF1, ovastacin and P22 repressor without affecting their function by the fused fluorescent protein. Therefore, the expression of the desired protein linked to their respective fluorescent protein with the help of a P2A peptide linker will take place. This linker will be self cleaved on its own after expression. Hence, we will get our desired protein and fluorescent protein.
Phase 1 was mainly designed to test the expression of proteins via individual cassettes of srtf1+mcherry, ASTL+sfgfp in our model organism Bacillus subtilis 168. We also designed a cassette in an attempt to improve the termination activity of an existing terminator using the strategy of making a double terminator.
We decided to express both srtf1+mcherry and ASTL+sfgfp cassette together in a single construct, which will result in the production of SRTF1 and ovastacin protein. The SRTF1 and ovastacin yielding cassettes are designed in such a way because they do not interact directly with each other. Therefore, they may not hamper the expression of each other. This will allow proper estimation of the production of both the proteins in Bacillus subtilis 168 by detecting fluorescence using a plate reader.
We planned to check the P22 repressor+Azurite cassette, independently. SRTF1 interacts with the P22 repressor cassette and inhibits P22 repressor protein expression. On other hand, the P22 repressor interacts with the ASTL+sfgfp cassette and inhibit ovastacin production. Hence, to estimate the expression of the P22 repressor protein without any hindrance, we planned to check it independently.
Additionally, we planned to check the expression of ovastacin A BBa_K3889022 which contains phosphomimetic for serine residue and ovastacin B BBa_K3889023 which contain phosphomimetic for serine and tyrosine residue. Here, we used phosphomimetics because we wanted to express them in bacteria, a prokaryotic organism. Bacterial cells cannot do a post-translational modification like phosphorylation because they lack the machinery to do so. These post-translational modification can play role in the structure which can also contribute to its functionality.
Simultaneously, We planned ovastacin B BBa_K3889023 purification in Bacillus subtilis 168 and ZP2 BBa_K3889029 expression and purification after transforming E.coli BL21 with plasmids. Which will be done by purifying plasmid from the clones that we got from Dr Satish K Gupta. After purification of both the protein, we planned to do an in-vitro cleavage assay with them. With this assay, we aimed to confirm the functionality of ovastacin B to cleave hZP2 protein.
In this in-vitro cleavage assay, ovastacin B was preferred as it contains phosphomimetic for serine and tyrosine residue. Hence, it has more chances of being purified successfully with its proper structure and functionality as it has an amino acid substitution that mimics the phosphorylated amino acid, i.e. serine and tyrosine, in the original structure of ovastacin.
Lastly, ASTL+sfgfp with signal peptide cassette was designed for checking the secretion of folded protein ovastacin in Bacillus subtilis 168 using 2 signal peptides, YwbN BBa_K3889051 and PhoD BBa_K3889050 .
From Phase 1, we would confirm the working of our individual cassettes and purify protein to perform an in-vitro cleavage assay. So, that we can conclude that ovastacin produced by bacteria can cleave hZP2. Hence we can move to Phase 2 of our wet lab workflow.
In Phase 2, we aimed to check the Protein-DNA interactions by combining two cassettes in Bacillus subtilis 168.
One of them was srtf1+mcherry and P22 repressor+Azurite cassettes with which we wanted to check the progesterone sensing. SRTF1 is expressed under a constitutive promoter SP126 BBa_K3889010 . In the absence of progesterone, the expression of SRTF1 represses the expression of the P22 repressor protein. Therefore, relatively higher red fluorescence will be detected in the plate reader as there will be a leaky expression of the Azurite blue fluorescence protein. While in the presence of progesterone, SRTF1 binds with progesterone. Due to which the binding of SRTF1 to the operator region of the P22 repressor cassette is hindered. This hindrance increases the expression of the P22 repressor protein. Therefore, in the presence of progesterone, two different fluorescence will be observed. In the plate reader, the red fluorescence of mCherry and the blue fluorescence of Azurite will be detected in the absence of progesterone due to leakiness.
Similarly, we planned to check the P22 repressor+Azurite and ASTL+sfgfp cassette expression and interaction of the P22 repressor with its operator region on the ovastacin cassette, respectively, by detecting fluorescence intensities. In this, the expression of ovastacin is repressed in the presence of the P22 repressor. Due to which only the blue fluorescence of Azurite will be detected as it is the reporter protein for P22 repressor expression.
From Phase 2, we will be able to show progesterone sensing and ovastacin regulation. This would be due to the confirmation of the interaction of both srtf1 and ASTL cassette with the P22 repressor cassette.
In Phase 3, we aimed to check the working of the genetic circuit as a whole in our model organism Bacillus subtilis 168.
Here we focus on the ovastacin regulation circuit, which includes constitutive expression of SRTF1 with its reporter mCherry regardless of the progesterone concentration. Now in the absence of progesterone, SRTF1 inhibits the expression of P22 repressor by binding to the operator region of the P22 repressor cassette. Thus, it allows the expression of ovastacin with its reporter sfGFP. While in the absence of progesterone, SRTF1 would bind to progesterone and thus, P22 repressor protein is expressed. Downstream to the P22 repressor, the Azurite reporter is being expressed too. This P22 repressor protein goes blocks the expression of ovastacin and sfGFP reporter in its downstream.
Thus, Phase 3 helps us give our proof of concept that ovastacin is being produced in a periodic and regulated manner to prevent fertilization due to successful progesterone sensing.
Along with the plan above, we designed a cassette to express ovastacin and hZP2 protein in Saccharomyces cerevisiae, a eukaryotic model organism. These proteins will be expressed under different inducible promotors to show in-vivo cleavage of eukaryotic protein i.e. ovastacin cleaving hZP2 in yeast as a model organism.
Here, we planned to produce ovastacin with the help of a galactose inducible system and ZP2 with the help of a copper inducible system in yeast. So that we could compare the structural difference in the protein produced in Bacillus subtilis 168 and Saccharomyces cerevisiae S288C. As if in Bacillus Subtilis 168, in-vitro cleavage assay does not give the desired result. Then we planned to do the supplementary experiment in the yeast i.e. in-vivo cleavage assay. By comparing the results and keeping in mind the differences in the structure of the protein being expressed by the respective organism. We will be able to postulate that for the successful cleavage to happen, post-translational modifications may be necessary and not having these modification can be the expected cause for not getting desired results. As these modifications would affect the functionality of the protein. And these modifications to happen, machinery is present in Saccharomyces cerevisiae S288C but not in Bacillus subtilis 168. Therefore, probably for ovastacin to functionally be active and cleave ZP2, it has to go through post-translational modifications.
To achieve the above aim, we need to design the experimental workflow.
Sequence Assembly is one of the most essential and significant processes of recombinant techniques and cloning. It allows multiple components of DNA to be physically linked together, bringing in existence the complete gene cassette.
Out of the multiple existing assembly techniques, Golden Gate Assembly is one of the more recently developed ones. Relying on the use of Type IIS Restriction Endonucleases and DNA ligase, this powerful and time-saving technique has the capacity to link together multiple parts and components parallelly, thereby enabling one-pot synthesis of the entire gene cassette.
As a supplement to Golden Gate assembly, the use of Biobrick assembly in the project ensured that we would achieve modularity while building constructs of more complexity.
Furthermore, another advantage was the introduction of mixed sites that are inherent in the Biobrick Assembly which served to separate our Level 1 constructs, thereby making the use of spacers redundant.
The use of two independent assemblies significantly increased the extent and modularity of our project.
Growing bacteria on an LB agar/YPD plate or liquid broth (LB/ YPD).
- Culture methods on solid growth medium are-
- Streaking - we get single colonies grown from a single parent bacterial cell to pick up for experiments like miniprep.
- Spreading- we get bacterial cells evenly spread on a plate. From which, we can take single colony or mass colonies according to the demand of the experiment.
- Culture method on liquid growth medium-
- Broth- This method helps us to grow and maintain the culture for further experiments. In this, the growth is viewed with the turbidity of the liquid.
It gives an idea about the growth rate of the bacteria by measuring OD across various time points. The rough idea is helpful in the experiments of competency and protein purification.
Cell competency and transformation
Cell competency is the common methodology for making cells competent to take up the extracellular DNA and the transformation is the natural process by which cells take up foreign DNA from the environment. We have different artificial methods to make cells competent and transform them with vectors like the chemical method and electroporation method. These techniques are used in modern recombinant DNA technology to transform the bacterial cells (e.g. E.coli DH5α for cloning experiments). Chemical methods and electroporation have their own protocols.
In cloning, the transformation of E.coli is used for the plasmid mass production while other model organisms like Bacillus subtilis and Saccharomyces cerevisiae act as an expression system for as per the demand of our experiments.
We did competency and transformation in E.coli DH5α and BL21 strains.
Miniprep and Nanodrop
Miniprep is the separation technique where we multiply the vector/plasmid within bacterial cells (here, in E Coli strains like DH5α, TOP10, DH10β) and purify it with a kit method or manually. This purified plasmid/vector can be used for further downstream processes like sequencing, restriction digestion and ligation.
Nucleic acid concentration is quantified by a device called a nanodrop spectrophotometer. It also provides information about the sample purity. We did quantification after the PCR amplifications and Minipreps.
We did various minipreps using kit methods for our plasmids like pRS426, pRS425, pRS424, pDR111, pDR110, pRSETb.
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Agarose gel electrophoresis and Gel extraction
Basically, the technique is used to analyse the results of cloning experiments. This analysis is done based on the relative separation of DNA fragments with respect to their mass and charge on the agarose gel. The percentage (w/v) of the gel is dependent on the DNA fragment sizes that we are dealing with.
Gel extraction is the technique in which we isolate and purify the desired DNA band on the gel using kit methods following the agarose gel electrophoresis.
Restriction digestion and PCR cleanup
Restriction digestion is a process in which DNA is cut at specific restriction sites by endonucleases like XbaI, NheI, BamHI, HindIII, SphI, etc. These reactions are carried out at particular temperatures to the enzyme in the thermocycler.
After performing the reactions in the thermocycler, we need to do PCR cleanup to remove impurities from the DNA samples. These impurities contain the master mixes, buffers, enzymes, etc., in the reaction.
Golden gate assembly
This is the in- vitro cloning method in which we can directionally assemble the multiple DNA fragments into one piece using type IIS restriction enzyme (e.g. BsmbI, BsaI) and T4 ligase.
Here, we design the multiple fragments and do a golden gate assembly to join those fragments to form one fragment, which will be the insert/GOI for the vector and then we can perform a ligation reaction.
Ligation reaction can be done separately or within the golden gate assembly reaction.
SpeedVac is the vacuum concentrator device in which temperature and vacuum are maintained in the chamber to evaporate the solvent and concentrate the sample. This is used in proteomics as well as in genomics and other various fields.
Patching and Colony PCR
Patching is the technique in which we use a sterile toothpick to pick up the colony and transfer it to the new agar plate.
One of the applications of Patching would be to get the colonies for the colony PCR.
Colony PCR is the method used to screen the colonies with the correct vector with GOI, which are grown on selective media.
Replica plating is the procedure by which we just make the secondary replica plates of the master plate.
We use it for the starch tests where we could grow the culture on amylase-containing secondary plates.
The starch test is a test that can be done to check the hydrolysis of amylose by amylase enzyme, which is produced by bacteria.
Here, we use the test to check the genomic integration at amyE site in the Bacillus subtilis genome.
There are different ways to do it. Furthermore, according to the experimental organism and protein of interest to be purified appropriate method is selected to lyse the cell. There are many ways that are Mechanical, Liquid Homogenisation, Sonication, Freeze-thaw, Manual grinding and simple chemical methods.
We standardised the method to use for our protein by doing a pilot experiment. We performed sonication along with a simple chemical method to ensure that we purify the functional protein. We were sceptical about our desired protein getting denatured due to the amplitude used during sonication. However, the final results confirmed that the sonication method of cell lysis is better for our protein.
Ni-NTA affinity protein purification
It is an affinity-based protein purification system for 6xHis-tagged recombinant proteins expressed in bacteria, yeast, insects and mammalian cells.
This method works on the principle of reversible interaction between a His-tagged protein and a specific ligand, i.e. nickel for the Ni-NTA system. It has high selectivity and high resolution.
In our project, we had 2 his-tagged proteins, ZP2 and ovastacin. Hence, we decided to use the Ni-NTA (IMAC- Immobilized Metal Affinity Chromatography) system to purify them.
It is the method to separate the proteins based on their molecular weight. It can be used for the rough idea about the weight of purified protein.
We used it for estimating the rough presence of our desired protein by checking the presence of bands near 90kDa for ZP2 and 22.5 kDa for ovastacin.
Immunoblotting is done to detect the desired protein with the help of primary and secondary antibodies. Primary antibodies go and bind to the desired protein on the blot. Then secondary antibodies are added, which goes and bind to primary antibodies. At last, the substrate is added, a chemical reagent that goes and reacts with secondary antibodies. After that reaction, it gives out a measurable signal which can be detected under UV light. Hence, it concludes that it has the desired protein.
We use 6X- His tag antibody as primary antibodies for our proteins ZP2 and ovastacin which were expressed in bacteria. And then we used horseradish peroxidase secondary antibodies. And then added substrate to develop the membrane and confirm the presence of our protein specifically.
Plate reader is also known as microplate reader. There are different types of microplate readers like Absorbance microplate readers, Fluorescence microplate readers, Luminescence microplate readers and Multimode microplate readers.
For our project, we are using a fluorescence microplate reader to measure the expression of sfGFP, Azurite and mCherry reporter proteins. Fluorescent molecule emission is filtered, collected, and measured with a better dynamic range than absorbance approaches in a fluorescence microplate reader. This fluorescent molecule emits light upon excitation by a higher energy light source. In this way, we do fluorescence intensity assays with the help of which we get to know the expression level of the promoter or protein. This fluorescence intensity assay can include single or multiple colours in one experiment with a diverse array of fluorophores.