What does gram negative bacteria mean?
Gram negative bacterias does not get stained during the gram staining method, while gram positive bacterias does. The difference between positive and negative is the difference cell walls, where gram positive have a thick layer of peptidoglycan that gets stained.
What different kind of sites does a typical plasmid vector have?
- Origin
- Selection marker
- Promoter
- Shine-Dalgarno (Ribosome binding site)
- N-terminal tag/fusion protein
- Multi-cloning site
- Restriction site
- Gene insert
- Terminator
What kind of information does a general vector contain regarding the specific protein that will be produced in the cell?
It contains the information for: 1. Where 2. When 3. How the specific protein will be produced in the cell
Can E. coli read genes of any organism?
???
What are introns?
Introns are the non-coding parts of a gene that is transcribed but then is removed during RNA splicing, before translation.
What three problems might prevent efficient expression of a foreign gene cloned in E.coli?
- E. coli can’t excise introns (a problem since E. coli don’t contain introns so the bacteria doesn’t have the necessary machinery for removing introns from transcripts)
- Premature termination of transcription (the sequences might be unproblematic in normal host cells but can act as a terminator in bacteria)
- Codon bias (almost every organism use the same genetic code but each organism has a bias toward preferred codons. This bias reflects the efficiency with which the tRNA molecules in the organism are able to recognize the different codons. If a cloned gene contain a lot of disfavored codons, the E. Coli might tRNA may encounter problems which means the amount of synthesized protein is reduced.) the result of this is that E. coli has difficulty translating the proline codons in a human gene.
What problem can be caused by secondary structure at the start of an mRNA?
The structure can interfere with the ribosome binding site by covering it.
How can you synthesize genes?
Algorithms can be used to optimize the sequence for expression in a given host and can then be synthesized chemically.
What are codon optimizations normally based on?
- tRNA abundance in the specific host
- Codon bias
- Avoid secondary mRNA structures
What is the level of expression dependent on?
The strength of the promoter.
What does a strong respectively weak promoter mean in terms of number of proteins translated?
A strong promoter leads to many transcripts which leads to many protein molecules translated.
A weak promoter leads to few transcripts which leads to a low number of protein molecules.
Can you regulate the promoter? What are these types of gene regulations called?
- An inducible gene
2. A repressible gene
How does a inducible gene work? An example
An inducible gene is normally of but when a regulatory chemical binds/enters, the gene switches on.
What is a repressible gene?
A repressible gene is a gene that is normally on but when a regulatory chemical enters/bind, the gene switches off.
Describe how the lac promoter works. Is it a inducible gene or repressible gene?
The lac promoter is the sequence that controls the transcription of the lacZ gene coding for beta-galactosidase. The lac promoter is induced by IPTG so when this chemical is present it switches on the transcription of the gene.
When lactose is present it binds to the repressor and the repressor let’s go of the operator and let the RNA-polymerase bind to the promoter and start the transcription.
What are hybrid protein generated for?
They are generated to facilitate gene expression and/or add new functionality to the protein.
How is a hybrid gene constructed and how is it synthesized?
The foreign gene is inserted after the start of an E. coli gene, which creates a hybrid gene that starts with E. coli and ends with the foreign gene, without a break into codons between the two different genes. The product of gene expression is therefore a hybrid or fusion protein, consisting of the short peptide coded by the E. coli reading frame fused to the amino-terminus of the foreign protein.
What four advantages does the fusion system have?
- Efficient translation of the mRNA produced from the cloned gene depends not only on the presence of a ribosome-binding site but is also affected by the nucleotide sequence at the start of the coding region. This is probably because the secondary structures resulting from intrastrand base pairs could interfere with attachment of the ribosome to its binding site. This possibility is avoided if the pertinent region is made up entirely of natural E. coli sequences.
- The presence of the bacterial peptide at the start of the fusion protein may stabilize the molecule and prevent it from being degraded by the host cell. In contrast, foreign proteins that lack a bacterial segment are often destroyed by the host.
- The bacterial segment may constitute a signal peptide, responsible for directing the E. coli protein correct position in the cell. If the signal peptide is derived from a protein that is exported by the cell, the recombinant protein may itself be exported, either into the culture medium or into the periplasmic space between the inner and outer cell membranes. Export is desirable because because it simplifies the problem of purification of the recombinant protein from the culture.
- The bacterial segments may also aid purification by enabling the fusion protein to be recovered by affinity chromatography. For example, fusions involving the E. coli glutathione-S-transferase protein can be purified by adsorption onto agarose beads carrying bound glutathione.
According to slide 14: - Stabilize mRNA molecules —> prevent degradation
- May have a signal peptide for correct targeting in the cell (secretion)
- Easier purification
What can leader peptides be used for?
To direct where the protein ends up.
What is inclusion bodies (IB)? Can it be a problem? How are they formed?
IB is a crystalline or paracrystalline deposit within a cell, often containing substantial quantities of insoluble protein. IB can be a major problem for production of recombinant proteins. IBs are formed by misfolded or unfolded proteins, which aggregates into non-functional protein clusters that are excluded by the cell’s quality control system.
What is protein folding often aided by?
Chaperones. (Hsp70 proteins in eukaryotes).
What is a non-integrative plasmid?
Plasmids that are able to multiply within the cell independently of the main bacterial chromosome.
What is an episome? What is an advantage of this?
An episome, or integrative plasmids, insert themselves into the bacterial chromosome to be able to replicate. Chromosomal integration are often more stable.
What are some reasons that long term continuous production mode may face some losses? What do we actually want?
- Loss of plasmids
- Down-regulated transcription/translation
We want a high concentration during the generation time. What we might get during “normal” production is concentration loss during the generation time.
