What can you do with microbial genome engineering?
- Study microbial cells, genotype-phenotype correlations, molecular mechanisms, metabolism, recombinant gene expression, enzymes etc.
- Develop industrial strains for commercial applications, for example microbial cell factories
What is the natural mutation rate for microbial cells?
Approximately 10^-10 base pairs per replication
How can the mutation rate be increased?
By radiation, chemicals, UV-light. Can be used to create libraries of strains with genome variation.
What is directed evolution?
It’s a process that alters between gene diversification and screening for or selection of functional gene variants.
What are some downsides with directed evolution?
- The expanded genetic space i randomly created
- Requires large scale screening
- New cell functions/properties are restricted by the original genome and its plasticity
Where are directed evolution a powerful technique?
In industrial strain development.
What is the general workflow for targeted genome engineering when using GMO?
- Design the gene
- Build the DNA –> Assemble the cloning cassette –> Transform into cells
- Identify cells with altered genome
- Experimental evaluation of cells with engineered genome
What is PCR?
PCR, polymerase chain reaction, is a technique you can use to detect a certain organism (the DNA/RNA of an organism).
What are the 4 components required for PCR?
- A DNA or RNA sample
- DNA-primers (a short single-stranded DNA that promotes the synthesis of a complementary strand of nucleotides)
- DNA polymerase (an enzyme that aids in the synthesis of a complementary strand of DNA
- Nucleotide solution mic (containing A,T,C,G that is used to build duplicated DNA strands)
What are the steps of PCR?
- Denaturation
- the solution is heated to at least 94 C which will break the hydrogen bonds of the original DNA sample and it will separate the DNA into single strands) - Annealing
- the solution is cooled to between 50 to 60 C which allows the primers and the DNA polymerase to bind to the individual strands of the DNA that was separated by the heat
- the nucleotides from the mixture solution will pair with the individual separated strands of DNA - Extension
- when the nucleotides has been paired with the individual strands, they will form a new complementary strand of DNA –> a new duplicated double-stranded DNA molecules has been formed from each of the single strands of the original sample molecule
These three steps are repeated about 35 to 40 times which will lead to a single short segment of DNA from one sample will be amplified to millions of copies.
- Analysis with electrophoresis
- after the PCR process is complete, the resulting amplified /replicated) segments generated can be compared to other nucleotide segments from known sources
- the nucleotide sequences can be placed next to known nucleotide sequences from humans, pathogens or other sources in a gel –> electrical current is run through the gel –> the various nucleotide sequences form band that resemble a ladder, according to their electrical charge and molecular size –> bands or ladder-like steps that migrate to the same levels in the gel shows the identity of the nucleotide sequence
What is an expression cassette?
??? An expression cassette is a component of vector DNA which consists of a gene and a regulatory sequence to be expressed by a transfected cell.
What is a cloning casette?
???
How are mutations introduced?
They are introduced by transforming cells with short sequences of ssDNA oligonucleotides (Synthetic oligos, max 200 bases)
Describe the steps of recombineering with single-stranded DNA.
- Mismatching/replace nucleotides
- resulting in: STOP codons, replace amino acids - Insert nucleotides
- frameshifts
- introduce amino acids - Delete nucleotides
- frameshifts
- remove amino acids
What does recombineering with ssDNA in E. coli require? Why does it require this?
It requires co-expression of bacteriophage gamma-Red ssDNA-binding protein beta. This prevents degradation of ssDNA.
How are allelic replacements achieved in E. coli? When does this happen?
By directing ssDNA oligonucleotides (oligos) to the lagging strand of the replication fork during DNA replication.
What kind of ssDNA oligos will be incorporated in the chromosome?
The ones with homology to the target.
What is homology?
When characteristics of different organisms look like each other and has the same evolutionary origin.
What is homologous recombination?
It’s a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of ds or ss nucleic acids (usually DNA in cellular organisms and RNA in viruses).
What’s the difference between plasmid and vector?
Plasmid is an extra-chromosomal element of mainly bacterial cells. Plasmid is a type of vector and is a circular double-stranded extra-chromosomal DNA molecule in some bacterial species.
Vector is a vehicle that carries foreign DNA molecules into another cell. Vectors are a self-replicating DNA molecules that acts as a vehicle for delivering foreign DNA into host cells
Plasmids can be used as vectors.
What is the difference between single-crossover integration and double-crossover integration?
??? Magnus
Single-crossover has one homology region while double-crossover has two.
How can you delete a gene by gene replacement?
By double-crossover integration. You replace the unwanted genes by replacing the chromosomal gene copy with another fragment.
What does the inserted DNA-fragment usually contain?
- Our wanted gene
2. Marker - that enables the selection of the double recombination event
Can you integrate both the marker and the wanted gene?
Yes.
