Our initial notion is to design a complete circuit that allows visible color change of different greyscale values, for us to measure the intensities of target molecules. First and foremost, we designed a module that allows stable expression of b-galactosidase. Bacterial endogenous ß-galactosidase can catalyze the chromogenic reaction of the colorless substrate x-gal or oNPG, and in combination with IPTG inducers, the chromogenic reaction will demonstrate a clear pattern of color change on different greyscale values.
The second module, the lysis module, is responsible for the visibility of our chromogenic reaction. We couldn’t observe the color change inside the cell’s cytoplasm through the naked eye, therefore, a lysis gene sequence comes into place. Phages contain a specific SRRz lysis gene in their gene sequence, the expression of which has the effect of lysing the bacterial cell wall. SRRz lysis gene is inserted into the bacterial plasmid, then, when the bacteria receive a signal from a specific molecule, the operon inserted upstream of the SRRz gene leads to its rapid expression, resulting in the lysis of the bacterial wall.
We selected two inducible promoters, the pBad/araC promoter (BBa_10500), also known as the arabinose promoter, and the copper-sensitive promoter (BBa_1760005), to activate our gene circuit. The RBS we used in our circuit is BBa_B0034, a relatively strong RBS used by many previous iGEM teams. Right after the b-galactosidase expression gene, the RBS is implemented again, followed by the SRRz lysis gene. Finally, we selected a double terminator BBa_B0015 to stop transcription. BBa_B0015 terminator is a double terminator, composed of BBa_B0010 and BBa_B0012. It is the most frequently used terminator in the community, emphasizing its high efficiency.
In the first version of our design, we succeeded in obtaining a colour change reaction. This proves that when either arabinose or copper ions were present in the solvent, the process was initiated and b-galactosidase was translated, and the SRRz lysis gene was successfully translated at the RBS. Hence, b-galactosidase flowed out to the surrounding solution and reacted sufficiently with X-gal to produce the colour change. However, we found that not only did the colour change fail to meet expectations, but that the gene circuit did not work efficiently, leading to bacterial death before sufficient b-galactosidase was produced.
After talking to our advisors, we found out that this was due to the fact that the lysis gene started working shortly after the activation of b-galactosidase production, so the bacteria died before adequate galactosidase could be produced. Through discussions among group members and arguments from the data, with the help of our advisors, we decided to apply an individual circuit for galactosidase expression to improve the effectiveness of the colour change.
For our new circuit, we adjusted the design of galactosidase expression. With the addition of a double-terminator and an inducible promoter, the galactosidase expression circuit became independent, allowing sufficient amount of galactosidase to be produced before the bacteria was triggered to death.
We have carried out the following improvements:
1. The addition of T7 promoter from T7 phage, which reacts specifically to the T7 RNA polymerase.
2. A double terminator was added prior to the lysis reaction to prevent RNA polymerase from continuing transcription. With the help of the terminator, RNA polymerase will be shedded from the DNA strand, thus terminating transcription.
The T7 promoter is used to translate galactosidase at the RBS, and then the reaction is stopped at the first terminator. For the lysis sequence, arabinose or copper ions are used as promoters to translate the lysis gene at the RBS, and when the cell is lysed, the reaction is halted by the terminator. With the addition of X-gal, a colour change is induced.
The results suggested that the lysis circuit works regularly, as the rapid decline of OD600 at 10^-5 mol/L indicates lysis of bacterial wall, which proves that our modified gene circuit could function normally and continue to work in a relevant context.
After a round of improvements to our detection system, our advisors collected samples of soil and river water from Dongdagou, Baiyin city, to test and verify the results of our cycle 2 gene circuit. The samples successfully undergo chromogenic reactions, demonstrating a series of blue, each in a different shade.
With the addition of the T7 promoter and the double terminator, the bacterium is able to produce galactosidase continuously before activation including the fermentation phase, the subsequent colour development with X is optimised and the bacterium is produced much more efficiently.
Our improved gene circuit functions more efficiently and conducts successful chromogenic reaction, as shown by the results of our experiments (See: Cycle 2-Test). For future improvements, we aim not only to maintain stability, increase efficiency and add new components to our gene circuit that aids regulation, but looking forward to innovative possibilities of inserting more operons into our bacterial plasmid. We will carry out more experiments, evaluating effectiveness of future modified plasmids, developing new concepts of multi-operon, and hence multi-functioning genetic sequence, balancing advantages to its trade-offs.
