Difference between revisions of "Team:IISER-Tirupati India/Design"

• Culture preparation-

Growing bacteria on an LB agar/YPD plate or liquid broth (LB/ YPD). 

• Culture methods on solid growth medium are- 

1. Streaking - we get single colonies grown from a single parent bacterial cell to pick up for experiments like miniprep.
2. 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-

1. 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. 

• Growth curve- 

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/vector 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, pRS425, pDR111, pDR110, pRSETb. 

• 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 iImpurities contain the master mixes, buffers, enzymes, etc., in the reaction.

• Golden gate Assembly and Ligation

This is the in- vitro cloning method in which we can directionally assemble the multiple DNA fragments into one piece using type 2s 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

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. 

In cloning, we can use the speedVac to concentrate the DNA samples with excess nuclease-free water so that the concentrated samples can be used for further downstream applications such as ligation reaction in golden gate assembly.

• 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.

Here, patching can be done 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 

Replica plating is the procedure by which we just make the secondary replica plates of the master plate. 

We used it for the starch tests where we could grow the culture on amylase-containing secondary plates.

• Starch test

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 used the test to check the genomic integration at amyE site in the Bacillus subtilis genome. 

• Cell lysis- 

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 method. 

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 

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 system to purify them. 

• SDS PAGE- 

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. 

• Western blotting-

Western blotting 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- 

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 linked protein. 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.