Team:Minnesota/Parts Characterization


Revision as of 16:46, 16 July 2009 by Bswiniar (Talk | contribs)
Home The Team The Project Submitted Parts Modeling SynBioSS Designer Parts Characterization Experiments

Parts Characterization

We chose five promoters that were included in the 2009 iGEM Kit to characterize. Using part BBa_F2620, which was characterized by a group from MIT in 2004, as a template, we characterized the following parts:

I14032Constitutive promoter classified as repressibleIPTGPrinceton 2004
J13002Two TetR binding sites and RBSaTcUT Austin 2005
I14015LasR, 3OC12HSL aTc regulated promoterLasR, 3C12HSL, aTcPrinceton 2004
K091101TTL AND gateIPTG, aTcDavidson Missouri-Western 2008
R0011Inverting regulatory region controlled by LacI; for comparison since already characterizedIPTGRegistry

We chose these parts because they had the same regulators as the promoters we examined in our project. Part 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 K081012, which consists of a strong RBS and GFP. This 'PoPS generator' takes 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 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 J13002, which already contained an RBS. This 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 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 pSB3K3, a low-medium copy plasmid with kanamycin resistance.

The 2009 iGEM Judging Criteria gives MIT's characterization of part 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:

  1. Transfer Function: the equilibrium relationship between the input and output
  2. Specificity: the ability of the devide to distinguish between its true input and similar inputs
  3. Response time: the time taken for the output to respond to a change in input
  4. Stability: how transfer function changes across multiple rounds of cell division and culture

In the Lab

Initially, we resuspended the DNA for the promoters, PoPS generator and GFP in water and transformed them into Top10 chemically competent cells. The plasmid backbone we used Then, we allowed the cells to grow overnight on the appropriate antibiotic plate based on the plasmid that the part was on. We picked colonies and inoculated liquid media. Once these cultures entered stationary phase, we prepped the plasmids using the QIAprep Spin Miniprep Kit for each promoter, the PoPS generator, GFP and the plasmid backbone (psB3K3) into which everything would be ligated. We quantified the purity of our DNA before performing polymerase chain reaction (PCR) to amplify the DNA we had from the plasmid prep.

Once the PCR completed, we usually ran some of the products out on an agarose gel to ensure that our DNA was the right size. We also sent some of our DNA to be sequenced and gratefully acknowledge the BioMedical Genomics Center (BMGC) at the University of Minnesota for their resources and expertise. The gel and sequencing helped us ensure that we had the correct plasmids.

We performed the restriction enzyme digest on PCR products and adjusted the reaction conditions based on the concentration of the DNA from the plasmid prep. This reaction ran for between 2 and 3 hours. We ran these products out on an agarose gel and excised the DNA with razor blades. Then, we purified the DNA using QIAquick PCR Purification.