Ampicillin → L-Hydroxyproline

A plasmid (pD4-α2, FIG. 68) encoding the gene for the carboxy terminal 219 amino acids of human Type I (α2) collagen with optimized E. coli codon usage as constructed in accordance with Example 14A fused to the 3′-end of the gene for glutathione S-transferase and under control of the tac promoter and containing the gene for ampicillin resistance was used to transform by electroporation proline auxotrophic E. coli strain JM109 (F-). Transformation cultures were plated on LB agar containing 100 μg/ml ampicillin. After overnight incubation at 37° C., a single colony from a fresh transformation plate was used to inoculate 100 ml of LB media containing 100 μg/ml ampicillin. This culture was grown to an OD600 nm of 0.5 and 100 μl aliquots transferred to 1.5 ml tubes. The tubes were stored at ˜80° C. For expression, a tube was thawed on ice and used to inoculate 25 ml of LB media containing 400 μg/ml ampicillin. After overnight growth at 37° C., a four ml aliquot was withdrawn, centrifuged, the cell pellet washed once with 1 ml of 2×YT media containing 400 μg/ml ampicillin, and the washed cells used to inoculate 1 L of 2×YT medium containing 400 μg/ml ampicillin. This culture was grown at 37° C. to an OD600nm of 0.8. The culture was centrifuged and the cell pellet washed once-with 100 ml of M9 medium (1× M9 salts, 0.5% glucose, 1 mM MgCl2, 0.01% thiamine, 200 μg/ml glycine, 200 μg/ml alanine, 100 μg/ml of the other amino acids except proline, and 400 μg/ml ampicillin). The cells were resuspended in 910 ml of M9 medium (1× M9 salts, 0.5% glucose, 1 mM MgCl2, 0.01% thiamine, 200 μg/ml glycine, 200 μg/ml alanine, 100 μg/ml of the other amino acids except proline, and 400 μg/ml ampicillin) and allowed to grow at 37° C. for 30 minutes. NaCl (80 ml of 5 M), hydroxyproline (7.5 ml of 2M), and IPTG (500 μl of 1 M) were added and growth continued for 3 hours. Cells were harvested by centrifugation and stored at −200° C.

1. customwere stored at ˜80° C
2. waitAfter overnight growth at 37° C.
3. customa four ml aliquot was withdrawn
4. washthe cell pellet washed once with 1 ml of 2×YT media
5. additioncontaining 400 μg/ml ampicillin
6. additioncontaining 400 μg/ml ampicillin
7. customwas grown at 37° C. to an OD600nm of 0.8
8. washthe cell pellet washed once-with 100 ml of M9 medium (1× M9 salts, 0.5% glucose, 1 mM MgCl2, 0.01% thiamine, 200 μg/ml glycine, 200 μg/ml alanine, 100 μg/ml of the other amino acids except proline, and 400 μg/ml ampicillin)
9. waitto grow at 37° C. for 30 minutes
10. additionNaCl (80 ml of 5 M), hydroxyproline (7.5 ml of 2M), and IPTG (500 μl of 1 M) were added
11. waitgrowth continued for 3 hours
12. customstored at −200° C.

ORD record

ord-19c02b5681664debbafa34c8e07994f2

Source dataset

uspto-grants-2005_08

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US06927287B1