Why

We first produced our gRNA plasmids in order to stop expression of targeted fluorescent proteins on our reporter plasmid.

How

Results

Why

How

Results

Why

How

Results

Why

The random ligation of different promoter-protein bricks is an experiment to test the viability of a brownian motion driven random ligation process. This random ligation is the basis for the unpredictability of a large scale key design process. The experiment hoped to produce a random mixture of fluorescent protein expression levels. This random mixture of multiple proteins in a single vector would then show the viability of the Key.coli restriction enzyme-ligation process for achieving unpredictability for keys.

How

The highest and lowest performing promoters were chosen to give the most easily visible result. Promoter E and Promoter 4. Promoter 4 gives a high expression of fluorescent proteins, as shown by our promoter library findings. The promoters were then attached to each reporter protein CFP, RFP and GFP via BSAI digestion and ligation (creating no scar sites) along with a terminator to form six "brick" variants. After amplification of the bricks produced via PCR, seven products combinations were ligated to a low copy backbone in the pattern of red, green and then blue (fluorescent protein) consistently through controlled use of the DCBA digestion site. In addition to a set of "random ligations". Each ligation has only one ligation slot (due to availability of restriction cut sites) per reporter type/colour, leaving random chance to produce a combination of all the possible variants in the random ligations where multiple promoters are available.

e= empty promoter, h= high expression promoter

These bricks were ligated with the backbone as follows:

This produced a control for each reporter fluorescent protein in isolation. It also produced a control for all reporters on either highest expression or lowest expression. This finally also produced a random set of colonies for isolation (after transformation) for comparison and test for true randomness.

Results

Why

For Key. coli to work as intended and not deteriorate we need need to things to occur:

1. The E. coli cells must be kept inactive so that nutrients is not depleted causing the transformed cells to die
2. The E. coli cells must be able to be activated after inactivation to allow the fluorescent genes to be expressed to give the key its unique fluorescent code which will allow access to the appliance.

How

To accomplish this, we chose to freeze dry the cells within the key. Click here for out protocol for freeze-drying cells.

Results

Figure 1: Graph of strong promoter 4 and weak promoter 1 transformed cells RFP fluorescence assay after freeze-drying revival two weeks and three weeks after samples were freeze dried.

In our results for freeze-dried cell revival, seen in Figure 1, we can show that storage temperatures do not have an effect on the revival of cells. For strong promoter 4 (SP4), at timepoint of 4 hours and 6 hours the storage temperature does seem to have a negative affect on the relative RFP fluorescence. However our sample size is very small meaning further assays would be needed to confirm this.

Why

How

Results