Protein Expression Prokaryotic cells - E. coli Lycopene

Protein expression refers to the techniques in which a protein of interest is synthesized, modified or regulated in cells. The blueprints for proteins are stored in DNA which is then transcribed to produce messenger RNA (mRNA). mRNA is then translated into protein. In prokaryotes, this process of mRNA translation occurs simultaneously with mRNA transcription. In eukaryotes, these two processes occur at separate times and in separate cellular regions (transcription in nucleus and translation in the cytoplasm). Recombinant protein expression utilizes cellular machinery to generate proteins, instead of chemical synthesis of proteins as it is very complex. Proteins produced from such DNA templates are called recombinant proteins and DNA templates are simple to construct. Recombinant protein expression involves transfecting cells with a DNA vector that contains the template. The cultured cells can then transcribe and translate the desired protein. The cells can be lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic protein expression systems are widely used. The selection of the system depends on the type of protein, the requirements for functional activity and the desired yield. These expression systems include mammalian, insect, yeast, bacterial, algal and cell-free. Each of these has pros and cons. Mammalian expression systems can be used for transient or stable expression, with ultra high-yield protein expression. However, high yields are only possible in suspension cultures and more demanding culture conditions. Insect cultures are the same as mammalian, except that they can be used as both static and suspension cultures. These cultures also have demanding culture conditions and may also be time-consuming. Yeast cultures can produce eukaryotic proteins and are scalable, with minimum culture requirements. Yeast cultures may require growth culture optimization. Bacterial cultures are simple, scalable and low cost, but these may require protein-specific optimization and are not suitable for all mammalian proteins. Algal cultures are optimized for robust selection and expression, but these are less developed than other host platforms. Cell-free systems are open, free of any unnatural compounds, fast and simple. This system is, however, not optimal for scaling up.

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Found 3 matching solutions for this experiment

pBAD–LCY

Martin Lohr, Institut für Allgemeine Botanik, Johannes Gutenber

Upstream tips	Protocol tips	Downstream tips
	Double transformants were grown in LB medium under induction with 0.2% arabinose and selection with chloramphenicol and ampicillin at 28°C and 250 rpm to an OD600 between 0.5 and 1.0.	Then, 10 ml culture per sample were harvested by centrifugation (5000 g, 4 min, 4°C), the supernatant completely removed and the cell pellet stored at −20°C until further analysis.

Protocol tips
Double transformants were grown in LB medium under induction with 0.2% arabinose and selection with chloramphenicol and ampicillin at 28°C and 250 rpm to an OD600 between 0.5 and 1.0.
Downstream tips
Then, 10 ml culture per sample were harvested by centrifugation (5000 g, 4 min, 4°C), the supernatant completely removed and the cell pellet stored at −20°C until further analysis.

Publication protocol 1 Relevant paper

pACLYC

Heng-Phon Too, Chemical and Pharmaceutical Engineering, Singapor

Upstream tips	Protocol tips	Downstream tips
	Four colonies were picked from agar plates, inoculated into 2xPY medium (20 g/L Peptone, 10 g/L Yeast extract, and 10 g/L NaCl, pH = 7) containing 34 μg/mL Chloramphenicol and 100 μg/mL Ampicillin, and incubated overnight. Ten micro-liter aliquots of overnight grown cell culture were inoculated into 1 mL 2xPY medium in 14 mL BD Falcon™ tube. Cells were grown at 37°C with shaking (300rpm) till OD595 reached the range of 0.5~1.0. The cells were then induced with various concentrations of L-Arabinose/IPTG and grown at 28°C or 37°C for indicated time before collected for RNA/protein extraction or Lycopene assay.

Protocol tips
Four colonies were picked from agar plates, inoculated into 2xPY medium (20 g/L Peptone, 10 g/L Yeast extract, and 10 g/L NaCl, pH = 7) containing 34 μg/mL Chloramphenicol and 100 μg/mL Ampicillin, and incubated overnight. Ten micro-liter aliquots of overnight grown cell culture were inoculated into 1 mL 2xPY medium in 14 mL BD Falcon™ tube. Cells were grown at 37°C with shaking (300rpm) till OD595 reached the range of 0.5~1.0. The cells were then induced with various concentrations of L-Arabinose/IPTG and grown at 28°C or 37°C for indicated time before collected for RNA/protein extraction or Lycopene assay.

Protocol tips

Four colonies were picked from agar plates, inoculated into 2xPY medium (20 g/L Peptone, 10 g/L Yeast extract, and 10 g/L NaCl, pH = 7) containing 34 μg/mL Chloramphenicol and 100 μg/mL Ampicillin, and incubated overnight. Ten micro-liter aliquots of overnight grown cell culture were inoculated into 1 mL 2xPY medium in 14 mL BD Falcon™ tube. Cells were grown at 37°C with shaking (300rpm) till OD595 reached the range of 0.5~1.0. The cells were then induced with various concentrations of L-Arabinose/IPTG and grown at 28°C or 37°C for indicated time before collected for RNA/protein extraction or Lycopene assay.

Publication protocol 1 Relevant paper

pT7lacO2

Mark P. Styczynski, School of Chemical & Biomolecular Engineerin

Upstream tips	Protocol tips	Downstream tips
	For lycopene expression, freshly transformed DH10B colonies were inoculated in triplicate into 5 mL LB medium containing the appropriate inducer and grown at 37°C and 180 rpm for 18 hours. For time-course experiments, overnight cultures were diluted to an OD of 0.15 in 50 mL of fresh medium, the appropriate inducer was added, and the sample was aliquoted into culture tubes corresponding to different time points. OD and lycopene content were measured at the time of inoculation, every hour for six hours, and at 18 hours.

Protocol tips
For lycopene expression, freshly transformed DH10B colonies were inoculated in triplicate into 5 mL LB medium containing the appropriate inducer and grown at 37°C and 180 rpm for 18 hours. For time-course experiments, overnight cultures were diluted to an OD of 0.15 in 50 mL of fresh medium, the appropriate inducer was added, and the sample was aliquoted into culture tubes corresponding to different time points. OD and lycopene content were measured at the time of inoculation, every hour for six hours, and at 18 hours.

Protocol tips

For lycopene expression, freshly transformed DH10B colonies were inoculated in triplicate into 5 mL LB medium containing the appropriate inducer and grown at 37°C and 180 rpm for 18 hours. For time-course experiments, overnight cultures were diluted to an OD of 0.15 in 50 mL of fresh medium, the appropriate inducer was added, and the sample was aliquoted into culture tubes corresponding to different time points. OD and lycopene content were measured at the time of inoculation, every hour for six hours, and at 18 hours.

Publication protocol 1 Relevant paper

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