During the first steps of the design process, our iGEM team discussed what engineering aims our project wanted to achieve. It was an important first step in determining the goals for our research. We based our engineering aims on Endy's "Foundations for engineering biology," from 2005.
After determining our engineering goals and aims for this year's competition. Our team needed to do background research into quorum sensing to familiarize ourselves with the terminology going to be used for this competition. In addition to this, our team needed to reaffirm the 2016 ASU iGEM team's data collected from last year before going any further with designing our project. While confirming last year's team data, we discovered a major flaws while running their protocols. These receivers were showed poor expression and only a select few were good enough to characterize. In addition to this, the system these receivers were cloned into did not show a good cloning ratio. In fact, it was very difficult to clone these receivers. After this, we realized we need to re-evaluate these receivers to determine whether it was the system we were cloning them in was bad, or the receivers themselves that needed to be redesigned.
From this, it was determined that the receivers themselves were bad and needed to be redesigned this year. From this realization, our design process was re-evaluated and is represented in the flow chart below:
In our receiver design, there a multitude of parts attained that are already Bio-Brick verified. The table below shows the various parts from the BioBrick database used in our design. Promoters, ribosomal binding sites, terminators and green fluorescent protein were chosen. In addition to this information, the reason and description of how each works in our system is described.
|Receiver Part Names||Part Number||Description|
|Ribosomal binding site for GFP||BBa_B0032||Stronger relative strength compared to other Ribosomal Binding Sites|
|Green Fluorescent Protein||BBa_E0040||Green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP|
|Terminator for Green Fluorescent Protein||BBa_J61048||Terminates the expression of GFP|
|Promoter for Receiver Protein||BBa_J23100||Stronger promoter relative to the GFP promoter for stronger transcription|
|Ribosomal Binding Site for Receiver Protein||BBa_B0034||Ribosomal binding site for receiver protein|
|Terminator for Receiver Protein||BBa_B0015||Strong terminator to end transcription of receiver promoter; A combination of RBS BBa_B0010 and BBa_B0012|
|Sender Part Name||Part Number||Part Type|
|C12-HSL, N-(2-oxooxolan-3-yl)dodecanamide Sender-AubI||BBa_K2033000||Sender|
|Isovaleryl-HSL, 3-methyl-N-[(3S)-2-oxooxolan-3-yl]butanamide Sender-BjaI||BBa_K2033002||Sender|
|3-phenyl-HSL, (3S)-3-[(2-oxo-3-phenylpropyl)amino]oxolan-2-one Sender-BraI||BBa_K2033004||Sender|
|3OH-7-cis-C14-HSL, (Z)-3-hydroxy-N-[(3S)-2-oxooxolan-3-yl]tetradec-7-enamide Sender-CerI||BBa_K2033006||Sender|
|C8-HSL, N-[(3S)-2-oxooxolan-3-yl]octanamide Sender*-SinI||BBa_K2033008||Sender|
|Part Name||Part Number||Part Type|
|Part Name||Part Number||Improvement|
|F2620||BBa_F2620||Reciever that our team further characterized and improved by running induction plates and various sender AHL experiments. In addition, safety for degrading and disposing.|
 Endy, Drew. "Foundations for engineering biology." Nature 438.7067 (2005): 449
 “PCR Cycling Parameters—Six Key Considerations for Success.” Thermo Fisher Scientific, Thermo Fisher Scientific. Web. 20 Oct. 2017.