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 to form six "brick" variants. After amplification of the bricks produced, seven products combinations were ligated to a low copy backbone as controls, in addition to a set of "random ligations" These ligations have only one ligation slot (due to availability of restriction cut sites) per reporter type, leaving random chance to produce a combination of all the possible variants. e= empty promoter, h= high expression promoter
These bricks were ligated with the backbone as follows:
eRFP-eGFP-eCFP - Low expression in all reporters
hRFP-hGFP-hCFP - High expression in all reporters
hRFP-eGFP-eCFP - Low expression in GFP and CFP, but High expression of RFP
eRFP-hGFP-eCFP - Low expression in RFP and CFP, but High expression of GFP
eRFP-eGFP-hCFP - Low expression in RFP and GFP, but High expression of CFP
eRFP or hRFP-eGFP or hGFP-eCFP or hCFP - As both variants of each reporter brick available, every combination is possible.
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
Twelve colonies were picked from the pilot study of random ligation, which gave the following distribution:
