Team:Arizona State/Design

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.

Extending the natural function spectrum and expression of new genes
In vitro evolution- biological regulation on a genetic and biochemical level
Genetic circuit construction for Quorum Sensing
Creating a quorum sensing system and chassis

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:

Receiver Design Flowchart

Receiver Design Flowchart for Cloning

Receiver Design

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

Senders Used in Our System

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

2017 New Receiver Systems

Part Name	Part Number	Part Type
TraR	BBa_K2357028	Reciever
LasR	BBa_K2357000	Receiver

iGEM F2620 Improvement

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.

References

[1] Endy, Drew. "Foundations for engineering biology." Nature 438.7067 (2005): 449

[2] “PCR Cycling Parameters—Six Key Considerations for Success.” Thermo Fisher Scientific, Thermo Fisher Scientific. Web. 20 Oct. 2017.