Part:BBa_J100198:Experience

This part works as designed. thyA- cells that contain J100198 do not grow unless theophylline is added to the media. Growth rates are comparable with 0.3, 0.6 and 1.25 mM theophylline added to LB broth + 50 µg/mL ampicillin (to select for pSB1A8 plasmid) + 50 µg/mL kanamycin (to select for thyA- mutation). J100198 is an improvement over Part:BBa_J100136 which failed because it permitted growth in the absence of theophylline.
File:ThyA cells.png
thyA- E. coli cells are auxotrophs that cannot make thymidine and can only grow if provided extra thymine in the media (50 µg/mL in LB media). When given provided with an E. coli gene expression cassette, the cells divide rapidly and are typical size for E. coli. Historical side note, thyA- cells were how biologists first discovered that bacteria do not divide until the DNA is replicated and that the cells will continue to grow in size. Size was NOT sufficient to trigger E. coli to divide.

Photograph of thyA- cells that contain J100198 in pSB1A8 grown in LB broth + 50 µg/mL ampicillin (to select for pSB1A8 plasmid) + 50 µg/mL kanamycin (to select for thyA- mutation). From left to right: 0, 0.3, 0.6 and 1.25 mM theophylline.

OD₅₉₀ of thyA- cells that contain J100198 in pSB1A8 Part:BBa_J119043 grown in LB broth + 50 µg/mL ampicillin (to select for pSB1A8 plasmid) + 50 µg/mL kanamycin (to select for thyA- mutation) with added theophylline as indicated. The cells were grown overnight and measured then ext morning. n= 3 and error bars represent standard deviation.
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