XOR gate design involving Omp system

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Why are we designing a new XOR gate?

We found a couple of characteristics of the components used in the old design (found here), that made it difficult to construct a functional XOR gate. The problems were:

  1. The Lux receiver (K09100) was induced by direct administration of both 3OC6 and 3OC12. Furthermore, Lux receiver cells were also induced by Las sender (K091136) cells.
  2. pLasLux and Las receiver (K091146 and K091134 respectively) could not be induced by 3OC12 (or 3OC6).

The Omp system in nature

Before you look at the XOR gate design below, you may find it helpful to check out how the Omp system works naturally in E. coli (The Omp system in nature). However, note that the explanation provided on the linked page is much simplified and presents only the information required to understand this project.

XOR gate design and concept

XOR gate construct.jpg

XOR truth table.jpg

The above design, which utilizes both the Omp system and the Lux system (information about the Lux system here), should show XOR gate properties if the following assumptions are true:

  1. When both inputs A (high osmolarity) and B (3OC6), the activities of the two promoters should negate each other and no output should be produced.
  2. When only one input is present (either high osmolarity or 3OC6), the activity of the induced promoter should be able to reach the GFP gene despite the physical separation (the uninduced promoter) between the two components.

Testing the pOmpC promoter

Before ligating all the parts together to make the XOR gate depicted above, I will test the promoters by themselves to make sure that they behave according to our expectations and are suitable for making an XOR gate.

Characterizing pOmpC

Two sets of primers were used to clone pOmpC because there are 80 bases between the end of pOmpC and the start codon of OmpC that we do not know the function of. The region could contain an intrinsic RBS, even though this was not evident from simply comparing the sequence of those 80 bases to the consensus RBS sequence. As a result, we decided to clone a long and a short version of pOmpC then test those with downstream GFP and RBS-GFP respectively. The pOmpC promoter can be induced with high osmolarity (see The Omp system in nature). LB media has been proven to act as a 'high osmolarity' media (1), while 'low osmolarity' has been achieved with TY media (2).

Characterizing strength of pOmpC.jpg

Is there any backwards activity?

Backwards activity in either pLux or pOmpC would be detrimental to the design of the XOR gate because an output would still be produced if both inputs are provided.

The first test will be done with Tet resistance placed upstream of both the pOmpC and pLux promoters because Tet resistance has been found to be a sensitive way to measure backwards activity in promoters. If Tet resistance is expressed from the backward activity of pOmpC, then RFP will be used, as this is a less sensitive test for backwards activity.

Backwards activity pOmpC.jpg

Backwards activity pLux.jpg

Possible hash function?

If the XOR gate is successfully developed, I may implement it to make a simple hash function. A simple hash function is depicted here.