Brautaset Flashcards
A heterologous gene originating from a plant is to be expressed recombinant
in the bacterium Escherichia coli.
a. Describe genetic modifications needed to achieve this
b. You manage a) but you observe very poor expression of your
heterologous gene product; describe three different strategies you can
test to increase expression level
c. Your gene product is host-toxic; describe two different strategies that
can be done to overcome this problem
a) The genetic modifications needed to express a plant gene in an E. coli bacteria include removing the exons, as the bacteria does not have the necessary machinery to perform the pre-mRNA processing, using a promoter that is expressible in E. coli, include a terminator that is functional in E. coli, have an origin of replication that is compatible with the bacteria, a ribosome binding site, a fusion sequence for affinity purification, and a signal sequence for secretion.
b) To improve the expression level, multiple things can be attempted:
1. Codon optimization utilizing codon bias
2. Increase the plasmid number/gene dose
3. Change temperature and other growing conditions
4. Change the RBSs
5. Improve mRNA stability to increase the translation of a single mRNA
Express the gene only after the cells are grown if the production is inhibiting for the cell. This is done by using inducible promoters.
If the product is host-toxic, one way to solve it is to fuse it to a signal peptide that will lead to secretion after the product is produced, thus removing it from the intracellular part of the cell. Another way is to use inducible promoters to not express the product until the cells are grown, in case the toxicity interferes with cell growth.
Fusion technology is a technique with many different applications; describe
how this technique can be used to solve problems related to
a. Protein solubility
b. Protein purification
c. Protein detection
a) To improve protein solubility, one important aspect is to avoid the formation of inclusion bodies (IBs), multiple unfolded proteins coming together in insoluble complexes. To avoid this, the fusion of a chaperone gene to the GOI can be done. The chaperone will promote the correct folding of the protein, thus preventing IBs.
b) To make protein purification easier, one can fuse a signal peptide that promotes the secretion of the product, or some other gene product that makes it easy to isolate by affinity purification.
To make protein detection easier, the GOI can be fused to a reporter gene. This reporter gene could encode GFP,his6 tag, Flag tag, for example, or some other molecule that is easy to detect.
Plasmids are commonly used as expression vectors; describe similarities and
differences between the pET and pBAD plasmid vectors for induced
recombinant expression in Escherichia coli
The plasmid vectors pET and pBAD are a bit similar and a bit different.
pET - uses a promoter from the bacteriophage T7 and a Lac promoter from E. coli. The Lac promoter can be induced by adding IPTG, and the T7 RNA polymerase is under control of this promoter. LacI, the repressor for the Lac promoter, is bound by IPTG, thus releasing the repressor from the DNA.
pBAD uses arabinose as an inducer, along with the araBAD promoter. If arabinose binds to the araC regulator, a conformational change in AraC will affect its ability to bind to the operator sites near the araBAD promoter, and AraC will go from a repressor to an activator. It is this binding that determines whether RBA polymerase binding, and thus the transcription of the gene, can happen.
The similarities between these systems is that both GOIs will only be expressed upon induction, and they can thus be controlled. The difference is that the pET utilizes a foreign promoter which requires a foreign RNA polymerase to transctribe its gene, whereas pBAD uses only a regulat repressor.
Describe how mRNA stability of one particular gene can be affected by
translation efficiency of this gene in Escherichia coli
mRNA stability of one particular gene can be affected by translation efficiency because the mRNA is subjected to degradation by RNases in the cytosol. However, suring translation, a ribosome will bind to translate the mRNA. This ribosome shields the mRNA from the RNases, and can thus increase the life span. The stability of the mRNA can be improved by changing the RBSs into ones that do not have inhibitory secondary structure elements.
Industry use fermentors to scale up recombinant protein production. Describe
advantages and challenges by cultivating recombinant E. coli in fermentors
and describe why it is particularly useful to use inducible expression systems
in such reactors
Some challenges with fermentors is that the high cell density will confer extra stress to the cells, and selection against the recombinant cells can therefore happen. This can be solved by using inducable promoters, which will lead to the product being produced only after the cells have grown, thus not inhibiting the production. pET and pBAD are examples of these kinds of promoters, using adabidose and IPTG as inducers. Also, an advantage of the bioreactors is that it is easy to control the environment, by changing pH, temperature, nutrition, aeration, etc., and that a high density of cells would be able to produce a lot of product.
