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Team:Minnesota/Parts Characterization
From 2009.igem.org
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Parts Characterization
We chose five promoters that were included in the 2009 iGEM Kit to characterize. Using part [http://partsregistry.org/wiki/index.php/Part:BBa_F2620 BBa_F2620], which was characterized by a group from MIT in 2004, as a template, we characterized the following parts:
| Part | Description | Regulators | People |
|---|---|---|---|
| [http://partsregistry.org/Part:BBa_I14032 I14032] | Constitutive promoter classified as repressible | IPTG | Princeton 2004 |
| [http://partsregistry.org/Part:BBa_J13002 J13002] | Two TetR binding sites and RBS | aTc | UT Austin 2005 |
| [http://partsregistry.org/Part:BBa_I14015 I14015] | LasR, 3OC12HSL aTc regulated promoter | LasR, 3C12HSL, aTc | Princeton 2004 |
| [http://partsregistry.org/Part:BBa_K091101 K091101] | TTL AND gate | IPTG, aTc | Davidson Missouri-Western 2008 |
| [http://partsregistry.org/Part:BBa_R0011 R0011] | Inverting regulatory region controlled by LacI; for comparison since already characterized | IPTG | Registry |
We chose these parts because they had the same regulators as the promoters we examined in our project. Part [http://partsregistry.org/Part:BBa_K091101 K091101] was particularly interesting because it was one of the constructs of the Tet and Lac operators that we examined for our research. Since Tet and Lac are commonly studied operators in synthetic biology, we wanted parts that involved them to be well-characterized to ensure the viability of future research.
We decided to characterize these promoters by attaching them to part [http://partsregistry.org/Part:Bba_K081012 K081012], which consists of a strong RBS and GFP. This 'PoPS generator' takes [http://parts2.mit.edu/wiki/index.php/Abstraction_hierarchy_and_PoPS PoPS] (Polymerase Per Second) as an input and gives GFP as an output, allowing us to indirectly measure PoPS and characterize our parts.
The picture at the right, from [http://www.jbioleng.org/content/2/1/5 Shetty et al. Journal of Biological Engineering 2008] demonstrates how to combine standard biological parts to form a new composite part. In our case, the prefix part was each of our 5 promoters and these were digested with restriction enzymes EcoRI and SpeI. The suffix part was the PoPS generator in every case except part [http://partsregistry.org/Part:BBa_J13002 J13002], which already contained an RBS. This [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 RBS] is defined as efficiency 1.0 while the RBS contained on our PoPS generator, which has an efficiency of 0.6. We decided to characterize the entire part that [http://parts2.mit.edu/wiki/index.php/UT_Austin_2005 UT Austin 2005] submitted, which included their RBS. These suffix parts were cut using restriction enzymes XbaI and PstI.
We ligated these parts to make a composite BioBrick part in [http://partsregistry.org/Part:pSB3K3 pSB3K3], a low-medium copy plasmid with kanamycin resistance.
The 2009 iGEM Judging Criteria gives MIT's characterization of part [http://partsregistry.org/wiki/index.php/Part:BBa_F2620 BBa_F2620] as an exemplar of parts characterization. We gratefully acknowledge their pioneering work with parts characterization and hope that our work continues to maintain the high standard for characterization of parts.
Like MIT in 2004, we characterized our 5 promoters included with the 2009 iGEM kit in terms of:
- Transfer Function: the equilibrium relationship between the input and output
- Specificity: the ability of the devide to distinguish between its true input and similar inputs
- Response time: the time taken for the output to respond to a change in input
- Stability: how transfer function changes across multiple rounds of cell division and culture
In the Lab
Initially, we resuspended the DNA for the appropriate parts in water and transformed it into [http://openwetware.org/wiki/TOP10_chemically_competent_cells Top10 chemically competent cells]
