Experimental Results

Testing of Or as a Promoter

The backbone of the radiation sensor in our project relies on part BBa_K648029. This part includes the operating region Or and two promoters, one on each end of Or. In order to test the functional ability of the of the pr promoter we attached a part called mcherry, which has rfp controlled by the promoter. We used the TECAN in our lab to measure the fluorescence of our samples over time. The data collected while the samples were in exponential growth was used to determine a rate of expression of the rfp. Compared to the control, the rate of fluorescence was much higher in our test subject, about 4227 compared to 418 (fu/OD600nm). This suggests that the promoter on Or is functional.

Future Testing

As part of our reporter project focused on improving the part developed by Imperial College London iGEM 2010 (BBa_K316007) we began by characterizing their test part K316009. The data for this test, using the standard catechol assay described by their team here is show below.

This data shows that, while their part is capable of extremely fast conversion of catachol to 2-hydroxymuconate semialdehyde, it is also subject to extreme input sensitivity that leads to significant results of color production seen from both induced and un-induced cells.

Much of the additional work for our project that expands on this data is still underway and due to time constraints cannot be posted before wiki freeze deadline. More characterization and testing of all the parts we are actively building is on the way and should be accomplished by the Jamboree.See our project pages for more information.

Parts

Favorites

BBa_K648011: Standard 25-Ready Xyle Reporter

This is a mutated version of the Xyle reporter gene which encodes for the enzyme catechol-2,3-dioxygenase (metapyrocatechase), which converts catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde. This version of Xyle has been made compatible with standard 25 assembly methods by removing three restriction sites (two NgoMIV sites: at bp 315 and 486, as well as one AgeI site: at bp 837). These mutations were made synonymous with the original sequence and codon optimized for E. Coli. Because of the synonymous mutations, this gene can be easily used to create fusion protein parts. For more information on the Xyle gene and its uses as a reporter see part BBa_J33204.

BBa_K648029/BBa_K648034: Or Operator

This is the operating region for the Lambda switch that repressors C1 and Cro bind to. This region contains promoters prm and pr facing in opposite directions. It also contains operating regions Or3, Or2 and Or1. In the lysogenic state, the C1 dimer is bound naturally bound to the Or1, repressing promoter pr and production of cro. Once RecA cleaves enough C1 dimer the system switches to the lytic state. The Cro produced at the pr promoter then binds to the Or3 site inhibiting the prm promoter. For a more complete description of the Lambda system read the book "A Genetic Switch" by Mark Ptashne. We used Or in creation of the lambda system, as part of our project to create a sensor for DNA damage in bacteria due to radiation.

BBa_K648123: RecA (Mutant)

RecA is a protein used by E. coli to repair and maintain DNA as well as perform homologous recombination. Many normal laboratory strains of E. coli like DH10B are mutants that code for an inactivated form of RecA known as RecA1. RecA1 is deficient in all known function of the RecA protein specifically in ATPase activity, binding with DNA in the presence of ATP, and changing conformation in the presence of ATP as well as repressor cleavage. In order to restore RecA1 to RecA and to reduce its recombinant activity while maintaining its proteolytic activity, a series of mutations were made.

One such mutation was made at amino acid 160, this adenine residue needed to be mutated to guanine to change RecA1 to RecA and restore its functions. In order to use the standard bio-bricking techniques we also had to remove naturally occurring enzymatic restriction sites. The Pst1 site CTGCAG needed to be converted to CTGCAA. Then the EcoR1 site GAATTC needed to be converted to GAATTT.

We also focused on how to mutate RecA so that it would be deficient in recombinant activity. After literature research we identified two amino acids in the RecA sequence that were known to affect RecA’s ability to perform homologous recombination. These sites were Arginine 243 and Lysine 286. Research suggested that changing Arginine 243 to Glutamine and Lysine 286 to Asparagine would remove recombinant activity from RecA.

List

Collaboration

NYC Wetware

Throughout the summer we connected with other iGEM teams interested in radiation-related projects. The NYC Wetware team in particular was studying the effects of genes cloned from the organism Deinococcus radiodurans on radiation resistance in E.coli. We were given the honor of helping them characterize one of their strains TS TR, a strain capable of producing the molecule trypanothione.

Using the gamma facilities provided by the Radiation Science and Engineering Center at Penn state we were able to carry out tests of gamma irradiation of their strain and subsequently measure cell viability.

Aliquots of culture (5ml each) inoculated with the TS TR strain were added to tubes which were irradiated by a cobalt-60 gamma source at a dose rate of 25 gys/min for total doses ranging from 0-800 gys. After irradiation 0.02 ml of the irradiated sample was inoculated into 5 ml of SOB media. The OD₆₀₀ was measured 12 hours later. The results are shown below.

Sevilla iGEM Team

We were also fortunate enough to have one of our team members, Lauren Rossi, visit the Sevilla 2011 iGEM team while traveling in Spain. She was able to meet the team, swap lab tips, techniques, and gossip, and even got a few members of their team to give interviews for our human practices video Check out their contributions and more on our human practices page!