Internship Progress Report

By Kailash Jayachandran 16INT0001

Background

Student at Pennsylvania State University (Class of 2019). Majoring in Biochemistry & Chemical Engineering. Did research in Environmental engineering. Currently a student mentor. Primarily interested in research & have a passion for it. Doing an internship at VIT to further enhance myself. Working in the labs of Dr. Karthikeyan, Dr. Gothandam, Dr. Rasool

& Dr. Babu at SBST. Learning various techniques under the tutelage of research

scholars.

List of Techniques

Inoculation & Streaking of cultures- Quadrant & Zig- Zag streaking Polymerase Chain Reaction (PCR) Agarose Gel Electrophoresis Principles of HPLC Bacterial Genomic DNA isolation Bacterial Plasmid DNA isolation SDS PAGE Immunofluorescence Western Blotting Principles of ELISA

Inoculation & Streaking of Cultures Generally used to grow specific bacteria in a nutrient broth This is generally done to isolate colonies by specificity. Factors of growth- Type of agar, conditions & composition of broth. Microbiological techniques- Sterilisation, aseptic techniques,

inoculation and incubation. Streaking techniques- Quadrant & Zig- Zag streaking

Quadrant Streak Plate Streak

Polymerase Chain Reaction (PCR) PCR is a technique which is used to amplify DNA & generate

millions of copies of that specific DNA sequence. It has 3 steps- Denaturation of the double-stranded DNA into a

single-stranded molecules; annealing of the primers to the specific area of interest and an extension phase.

Required materials- DNA substrate, forward & reverse primer, Taq Polymerase, dNTP’s, Buffer solution and divalent cations ( Mg2+ ).

Three types of PCR used in the lab- Gradient, Real time & Reverse transcriptase PCR.

Used in medical & biological research (genetic diseases & forensics)

PCR full process

Agarose Gel Electrophoresis

Separation of the PCR product/ DNA strands is required. This is done by using an electric field to move DNA towards the

anode & they move accordingly based on length (Charge/size). Required materials- Agarose solution, TAE buffer & Ethidium

Bromide. Thus, dyed samples are added to the casted gel. Visualization is done via UV transiluminator or Gel doc.

Visualization via UV

Visualizing gradient PCR product under UVGel Electrophoresis

Principles of HPLC HPLC is used to separate, identify & quantify components dissolved

in a liquid solvent with a high analytical resolution. HPLC pumps the sample in the mobile phase at high pressure

through a column filled with chromatographic material ( stationary phase).

Gas stream of helium & nitrogen help in facilitating sample transport.

So, compounds present in trace amounts can also be measured. Sample retention time will vary depending on the interaction

between the stationary phase, the molecules being analyzed, and the solvent, or solvents used.

Interaction between both columns is at different rates. So, this will impact the exit time of the sample.

HPLC Machine

HPLC Mechanism

Bacterial Genomic DNA Isolation Bacterial cells are grown in a medium till they reach log phase & are

centrifuged. Then, resuspension is done by adding lysis solution & Proteinase K is

used to degrade the gram negative bacterial cell wall. Cell lysis is followed & ethanol is added for binding with the lysate. The lysate is loaded onto a spin column (DNA binds to silica gel

membrane) & centrifugation is done with the flow through being removed.

Pre wash & wash is done using the respective solutions with the flow through discarded after centrifugation.

This removes trace amounts of salt and protein contaminants. The DNA is eluted using an elution buffer & the eluate is stored. Thus, these are the steps to isolating bacterial genomic DNA.

Bacterial Genomic DNA Isolation

Visualizing presence of Genomic DNA

Bacterial Plasmid DNA isolation The harvested bacterial culture is resuspended using a

resuspension solution for disturbing the pellet Then, the lysis solution is added to lyse the cells & the

neutralization solution is added leading to a cloudy solution. Centrifugation is done & the supernatant lysate is added to the

spin column (DNA binds with silica column in high salt concentration) with the sample being centrifuged again.

The flow through is discarded in the centrifugation processes. Pre wash & wash is done using the respective solutions with the

flow through discarded after centrifugation to remove contaminants.

The DNA is eluted using an elution buffer & the eluate is stored. Thus, these are the steps to isolating bacterial plasmid DNA.

Bacterial Plasmid DNA Isolation

Visualizing presence of Plasmid DNA

SDS PAGE SDS PAGE is a technique used to separate proteins by electrophoresis. Apart from charge & size, molecular mass plays an important role in

electrophoretic mobility. Preparation- Milli-Q water, Acrylamide, Bisacrylamide, SDS, APS &

TEMED. Acrylamide acts as the gel medium for protein movement &

Bisacrylamide helps in crosslinking acrylamide molecules. APS acts as a catalyst for polymerization & is a free radical agent &

TEMED enhances this effect. SDS helps in denaturation of proteins & imparts negative charge to

them. Thus, SDS- PAGE helps us to separate proteins effectively & is used in

techniques like western blotting as well.

Visualization of results

Gel Apparatus

Immunofluorescence Immunofluorescence is a microscopic technique using a fluorescent

microscope for microbiological samples. The specificity of antibodies are used to target the antigen using a

fluorescent dye to specific bio molecular targets which is visualized. Blocking using the Blocking buffer is done as BSA attached to

membrane with no target protein attached which eliminates false positives.

Then, incubation is done by adding the primary antibody & secondary antibody which is linked to DAPI & Alexa Fluor Stain.

This process is possible due to the constant (structure) & variable region (antigen acceptor) which allows various secondary antibodies to act on it.

Thus, we visualize the samples using a fluorescent microscope to obtain the images.

DAPI Stain

Alexa Fluor Stain

Western Blot Western blot is an analytical technique used to detect specific

proteins in a particular sample. First, the process begins with SDS-PAGE which helps to separate

the proteins by denaturation & electrophoretic movement. For the proteins to be more accessible to antibody detection, we

need to transfer the separated proteins to nitrocellulose or PVDF membrane.

By capillary action & electro blotting, this process occurs. Blocking is done by adding 3-5% BSA & 0.1% Tween-20. The diluted protein attaches to the membrane with no target

protein attached which eliminates false positives during antibody addition.

Incubation is done by adding a primary & secondary antibody. Visualization is doe by chemiluminescent detection.

Radiographic visualization

Sample Results

Western Blot Procedure

Principles of ELISA ELISA is a quantifying technique for detecting & measuring

proteins, peptides, antigens & antibodies. Adsorption of the unknown antigen to the assay plate allows the

addition of the antibody which facilitates antigen- antibody interaction.

A detection enzyme is generally linked to the antibody. E.g. Horseradish peroxidase and Alkaline phosphatase.

Detection is done by assessing enzyme activity by adding a substrate to give a measurable product.

Thus, non-binding material are generally washed away making it a powerful tool.

Some types of ELISA

Apparatus

Acknowledgments

I would like to convey my thanks to VIT University for the opportunity & support which made this experience possible.

The faculty of School of Bio sciences & Technology were very helpful & I sincerely thank them for their guidance.

Special thanks to Dr. Karthikeyan, Dr. Rasool, Dr. Gothandam, Dr. Babu & Dr. Ramalingam for making this possible.

The PHD scholars & research fellows patiently accommodated & taught me the techniques & instilled passion and knowledge in me.

Special thanks to Mr. Pavan Kumar, Mr. Ganesan, Ms. Madhuri, Mr. Dinesh, Mr. Prasanth, Ms. Shanthini, Mrs. Ajitha, Mrs. Pooja, Ms. Indira, Mr. Chaitanya, Mr. Srinivasan & Mr. Manoj.