To me, there’s a special beauty known only behind the curtain of the microscope room, in the faint red light of the darkroom, in the depths of the cave when nobody else is around.

I just pay attention to papers and tools and spread their insights when I can. Most bacterial eng-experienced degree holders wouldn't see any problem in your design.

To reduce selection pressure to break promoters, it's good practice to clone in a strain that expresses the TF to keep the promoter repressed. That or have the same construct also express it.

You might just need to change the second operator to lacOsym. Oh and there's a few papers that observe a periodicity to leak across spacings of operators. LacO works best when spaced in multiples of 11 bp center to center, so that the LacIs end up on the same face/helical angle of the DNA.

Also your lac operators aren't the minimal ones; needless flanking bases. Look up lacOsym, the stronger minimal symmetric lacO. Shorter the repeat, lower the recomb rate. Check with EFM Calc.

I fixed it using EFM Calc, making 2 small changes around/within the operator at neutral positions based on operator consensus. It made it stable over days of turbidostat growth. No change in leak.

I've seen this happen in four people's hands with the P.CymRD promoter in the Voigt Lab Marionette collection. It has two identical large operators. Barrick Lab's EFM Calculator is a great data-driven tool for predicting recombination. Promoters are especially prone because of selection pressure.

B.

I've been looking forward to someone figuring out a design for a Type II secretion tag. Some sort of peptide or domain that binds the pseudopilus and targets the cargo protein for secretion.

Some Gboard emoji combinations I'll be using to express my feelings in the lab, a thread

Here are some syn bio–themed file/folder icons I made a many years ago (.ico). github.com/shyambhakta/...