In Experiment 7, plasmid pETBlue-2 / hCA2 was used to transform E. coli TunerTM(
ID: 1012891 • Letter: I
Question
In Experiment 7, plasmid pETBlue-2 / hCA2 was used to transform E. coli TunerTM(DE3)(pLacI) cells (a map of pLacI is shown below; the cam gene encodes a chloramphenicol resistance protein). Why was pLacI constructed from pACYC184 (https://www.neb.com/~/media/NebUs/Page%20Images/Tools%20and%20Resources/Interac tive%20Tools/DNA%20Sequences%20and%20Maps/pACYC184_map.pdf) rather than pETBlue-2?. Why was it important that pACYC184 carried a gene that allowed resistance to the antibiotic chloramphenicol rather than resistance to ampicillin? .
Explanation / Answer
The pdf file from New England Biolabs cannot be accessible to me. It is showing error and may it could be due to unauthorized login. Hence your first question cannot be answered by me, sorry for that.
A general trend for such situations is as follows.
All of the pET translation vectors contain translation stop codons in all three reading frames following the cloning and tag regions as well as a downstream T7 transcription terminator. The terminator is not necessary for the efficient expression of most proteins, but note that some pET plasmids contain the gene for ampicillin resistance (bla) in the same orientation as the target gene. If the T7 transcription terminator is removed during cloning, IPTG-dependent accumulation of -lactamase (Mr 31.5 kDa) is usually observed along with the target protein, due to efficient read-through transcription by T7 RNA polymerase. Ampicillin selection tends to be lost in cultures because secreted -lactamase and the drop in pH that accompanies bacterial fermentation both degrade the drug.
Use of ampicillin as a selective antibiotic requires special care because -lactamase is made in substantial amounts and is secreted into the medium, where it can destroy all of the ampicillin. In addition, ampicillin is susceptible to hydrolysis under acidic media conditions created by bacterial metabolism. This means that a culture in which the cells carry an unstable plasmid will be growing under ampicillin selection only until enough -lactamase has been secreted to destroy the ampicillin in the medium. From that point on, cells that lack plasmid will not be killed and will begin to overgrow the culture.
Another way of providing additional stability to target genes is to express them in host strains containing a compatible chloramphenicol-resistant plasmid that provides a small amount of T-7 lysozyme, a natural inhibitor of T7 RNA polymerase. T7 lysozyme is a bifunctional protein: it cuts a specific bond in the peptidoglycan layer of the E. coli cell wall, and it binds to T7 RNA polymerase, inhibiting transcription. T7 lysozyme is provided to the cell from a clone of the T7 lysozyme gene in the BamH I site of pACYC184. The cloned fragment also contains the f 3.8 promoter for T7 RNA polymerase immediately following the lysozyme gene. The Rosetta™ strains supplement tRNAs rarely utilized in E. coli on a chloramphenicol resistant plasmid (pACYC backbone) compatible with pET vectors.
Updated answer for question 1:
Tuner™(DE3) is a DE3 host strain and allow variation of the expression level simply by varying the concentration of IPTG added to induce expression. These strains contain the lacY1 mutation eliminating the active transport of lactose into cells via lac permease. Therefore, these strains are less sensitive to lactose in the media and IPTG induction results in a more uniform entry into all cells in the population.
The subjection of Escherichia coli cells to heat shock (45°C, 15 min) causes the intracellular denaturation and aggregation of proteins. The aggregated proteins form a fraction, denoted S, which can be separated by sucrose density gradient ultracentrifugation. In the wild-type cells, the S fraction is detectable for only 15 min after the temperature change from 30°C to 45°C, and it disappears during the 10 min following incubation of the bacterial culture at 37°C. This disappearance depends on the effectiveness of the mechanism of heat shock response. In the rpoH mutants (unable to induce the heat shock response) the S fraction was found to be stable. Experiments performed with mutants that lack activity of certain chaperone proteins (DnaK, DnaJ, GroES or GroEL) revealed that dnaK and dnaJ mutations stabilized the S fraction, while groES and groEL mutations caused its incomplete removal. On the other hand, overexpression of DnaK/DnaJ or GroEL/GroES proteins completely prevented the transient appearance of the S fraction in the wild-type strains. The process of elimination of the heat-aggregated proteins was also retarded in protease-defective mutants (clpA, clpB, clpAP, clpXP, clpBP, and htrA63) and did not reach completion after one generation time (45 min). This demonstrated the biological role of Clp and HtrA proteases. Moreover, proteins included in the S fraction may be considered natural substrates for these proteases. Supposedly, the removal of the S fraction involved renaturation and proteolysis.
pACYC184 is a ColE1-type replicon, in the cells causes a 10-min delay in S fraction removal. pACYC184 carries chloramphenicol-resistance (cat) gene, a high-level expression of gene encoding protein that confer resistance to chloramphenicol, but not genes encoding -lactamase and aminoglycoside phosphotransferase (APH), is responsible for the decreased efficiency of removal of the S fraction from the cells. overexpression of cat from plasmid pLacI gives even stronger effects on S fraction removal than those observed with plasmids bearing this gene under its natural promoter. The effects on the removal of the S fraction arise from expression of the antibiotic-resistance gene rather than from addition of the antibiotic. Higher stability of the S fraction in cells expressing some antibiotic-resistance genes from plasmids might suggest that production of heterologous proteins in E. coli may result in lower efficiency of removal of denatured proteins, perhaps due to overloading of the S fraction.
The overproduction of a heterologous protein (T7 RNA polymerase) leading to the overload of the S fraction and the reduction of a pool of free chaperones that are normally available for assisting protein folding or protein disaggregation. Perhaps, the same mechanism is responsible for a delay in S fraction removal from cells expressing some antibiotic-resistance genes whose products are accumulated in the cytoplasm (e.g. CAT). According to this proposal, overproduction of proteins localized in the periplasmic space (e.g. -lactamase, aminoglycoside 3-phosphotransferase) had no effect on the removal of heat-aggregated proteins.