Biotechnology Flashcards
How is biotechnology implemented to our lives?
Food industry - Helps making lactose-free milk
Medicines - used to mass produce medicines such as penicillin and insulin
Agriculture - makes harvest more nutritious or have longer shelf life
Cellular vs Molecular biotechnology
Cellular - Does not need deep understanding of DNA and RNA
Molecular - Needs to understand how to manipulate cultures in a DNA and sometimes RNA level
What microbes are being used in biotechnology?
Virus, Archaea, Bacteria, Algae, Fungi
What function does viruses play a part in biotechnology?
Bacteriophage - type of virus that infects bacteria by attaching to them and uses their vectors to carry genes to the host
T4 ligase - Source of enzyme to join two DNA strands
Archaea in biotechnology
Used for their RNA polymerase as archaea lives in extreme environments meaning that their proteins are extremely thermostable, including the RNA polymerase. It can maintain activity in high temperatures
Bacteria in biotechnology
Its main role are as host cells, meaning the cells that carries the new recombinant DNA. It is good at cloning (making identical DNA) and expressing proteins to make high value products or enzyme
Algae in biotechnology
There is potential in using it as biofuels as it have a unique property of converting CO2 into hydrogen or ethanol, which are two components that are able to be used as fuels
Fungi in biotechnology
The use of mold can be made into antibiotics, anticancer synthesis and immunomodulators. Yeasts can also act as a host to express proteins and grows fast
E. coli vs Saccharomyces as host cells
E-coli - easy to tamper with the genes, can be a part of our normal flora, easy to extract genes, beneficial to digestion and immune regulation
Saccharomyces - better at expressing eukaryotic genes (since it is a eukaryote), generally recognised as safe (GRAS) which means that it can be digested
Plasmid
Little circle of DNA typically found in bacteria. Can be easily gained or lost from the cell as it contains optional extra information
Uses of plasmids
Common vector to introduce new DNA into a microbial cell
Origins of plasmids
We can isolate plasmids from bacteria whether the bacteria is taken from soil, water or the human body. Plasmids can swap DNA easily with other bacterium (horizontal gene transfer), because of this bacteria can evolve.
How to use plasmids
- Replicating features - allows it to replicate independently
- Selectable marker - lets plasmids be taken up by the bacterial cells by force
- Cloning site - used to insert the foreign DNA because it is a site recognised by restrictions enzymes where it can cut up that part of the DNA to insert a new DNA in
Cloning
Means to make identical organisms
3 ways to clone
- In vitro - using polymerase chain reaction (PCR) to replicate pieces of DNA
- Propagation - look back on Cavendish bananas
- Plasmid insertion - putting the DNA into a plasmid can make DNA be replicated
Tools needed for DNA cloning
- Copying DNA
- Cutting DNA
- Joining DNA
Amplifying/Copying DNA enzyme
Using the process PCR where we can copy DNA using a thermostable polymerase from the archaea
Cutting DNA enzyme
Restriction enzymes (endonucleases) are used to break down fragments of DNA (or mRNA). The same restriction enzymes needed to be used to cut the DNA and plasmids so that it recognises where the DNA should be at
Joining DNA enzyme
To join the DNA fragment and the plasmid together, need to use ligase such as T4 ligase
Steps to DNA cloning
- Digestion and Ligation
- Transformation and screening
- Product: GMO
First step to DNA cloning
Extract or amplify the DNA that we want (that produces the product that we want). Uses PCR to amplify/copy selected DNA. DNA is the ‘digested’ by cutting it into different pieces using a restriction enzyme and use the same restriction enzyme to cut the plasmid vector. Put the DNA fragment and plasmid together for ligation using the ligase to make a recombinant DNA
What is in the ligation mixture?
Contains recombinant plasmids, non-recombinant plasmids and non-ligated DNA
Why are there leftovers in the ligation mixture?
Because restriction enzymes and ligases are not 100% efficient so there will always be some starting plasma left over and bits of DNA that are not ligated
Second step to DNA cloning
Transformation and screening is needed to get only the recombinant DNA out of the ligation mixture. Uptake of DNA (Transformation) be done through electric shock, heat or conjugation (transfers itself into a cell). Pours the mixture into a selective agar plate which enables the plasmid-containing cells to grow. Screens the cells of interest (the one with recombinant DNA) either phenotypically (fluorescent) or genotypically (using PCR for sequence based screen).
Final product
GMO (genetically modified organism) is the final product. It is a recombinant gene carrying microbe.
What are the risks of GMO?
It usually depends what foreign DNA is added into the organism. Some people may be allergic to the DNA that is inserted into the gene however overall it is very safe. However, dealing with dangerous genes, it needs to be handles in a lab or physical containment level. It also begs the question on legal constraints around GMO. Another risk if there is poor handling in these organisms and is able to make their way to our normal flora, it can be extremely harmful for the human body, animals or environment. Commercial risks: understanding whether the public can accept it as a product. So in short, there are ethical and public considerations to be made for GMO
How is vaccine an example of a recombinant product?
Vaccines contain recombinant genes as the genes contained in vaccines are used to train the body’s immune system to recognise the target antigens. This type of vaccines contain genes of the target antigen
4 types of vaccines
- Live attenuated microbe - live microbe that will stimulate the immune system to respond
- Kill microbes - Kill the cells, may still be quite antigenic, can also stimulate immune system
- Antigen
- mRNA coding for antigens - stimulating your body by presenting the antigen
How does vaccine uses its genes to train the immune system?
It uses the antigen gene to express foreign protein (or antigen protein) in the body which is safe as this is only a surface protein of the virus (used to detect by immune system) that does not contain any other elements of the hepatitis virus
