Overall Aims:

To engineer the commensal E. coli, Nissle 1917 to absorb large volumes of glucose (and galactose).
This will enable the bacteria to function like a sugar sponge in the intestines sequestering sugar
and preventing it from being absorbed.
We will look at this in respects to a treatment for obesity and reducing calorific intake.

---

Biobricks used:

osmY (full sequence) Part:BBa_J45992:

• Full length sequence of the osmY stationary phase promoter.
  This promoter recruits RNAP with the sigmas haloenzyme which is expressed mainly during periods of low growth or starvation.
  It is also slightly activated by the sigma 70 exponential phase RNAP as well.
• We will use this to control expression of our mutant CRP so it is only expressed once the cells are fully grown and not during the growth of the Nissle.

---

New Biobricks:

CRP (cAMP reactive protein/peptide also known as CAP (cAMP activating protein/peptide)).

• WT CRP will be used mainly as a control to compare to the effects of the mutant CRP.
  CRP can only bind DNA when it is activated by binding to cAMP. cAMP levels are controlled by adenylate cyclase which itself is controlled by enzyme IIa part of the glucose uptake system in E. coli.

T127I/S128I Mutant CRP

• This mutant version of CRP is constitutively active regardless of the levels of cAMP.
  This means that it can induce glucose uptake regardless of the levels of cAMP in the cell and therefore the cells become glucose insensitive and absorb excessive volumes of glucose.

GlmS Riboswitch (from B. Subtilis)

• The GlmS riboswitch is found in most gram positive bacteria.
  It is found in the 3’-UTR of the GlmS gene which is responsible for increasing the levels of glucosamine-6-phosphate, which is used to make cell walls etc. Normally when GLcN-6P is present it binds to the riboswitch and causes cleavage and degradation of the mRNA.
  Glucose can also bind to the riboswitch but it prevents cleavage and allows transcription. We will use this to ensure that our gene is only expressed in high glucose conditions.

GlmS Promoter (from B. Subtilis)

• Normal GlmS promoter, this will be tested in parallel with the osmY stationary phase promoter and will be used to express the riboswitch and CRP.

GFP GlmS Riboswitch Fusion

• Reporter to analyse the effects of the riboswitch in different concentrations of glucose and to determine how much cleavage may occur in the intestines.

Mutant CRP GlmS Fusion

• Final product (+promoter) will be inserted into Nissle 1917.

---