(The following is a plan I was making to try to get pGKL1 into Escherichia coli. It did not work. -KG)

List of experiments needed

1) Is the pGKL1 replication machinery toxic when expressed in E coli.

2) Test the relative growth times of pGKLColi and pGLO with expression and without expression of proteins under the araBAD promoter

3) I assume that

1) Both the bacteria cells inherently grow at the same rate

2) Both strains have not recombined to pick up the resistance in any other ways then using the plasmid

4) Put in a liquid culture overnight of

1) pGLO without arabinose (neg)

2) pGKLColi without arabinose (neg)

3) pGLO with arabinose (neg)

4) pGKLColi with arabinose (variable)

5) E coli top10 cells without either (neg)


1) check the fluorescence of 1, 3, and 5 to find relative amounts of expression

2) Place 1,2,5 in flasks/plates without arabinose (1/100 dilution or other)

3) Place 1,2,3,4,5 in flasks/plates with arabinose induction (1/100 dilution or other)

4) Check the OD of each, and check the fluorescence of the controls and the pGLO plasmids every 20 minutes for 5 hours.

5) Compare and make graphs of each

5) If the growth curves of the induced pGLO and pGKLColi match each other, this shows that the pGKL1 machinery is non-toxic when expressed. If the growth is slightly stunted by induction of the pGKLColi machinery, it shows that it is toxic. If almost no growth is detected, then the pGKLColi machinery is very toxic. If no growth is detected, then the pGKLColi machinery are extremely toxic. If the pGKLColi machinery growth is slower than normal, but matches roughly matches the induced pGLO, then it means that the expression is not toxic because the expression is just slowing the growth.