Phactory Parts
Our parts collection consists of four basic parts and one improved composite
part. All parts were generated during our project. Some parts relate to RecBCD
enzyme & its inhibition and some to the translation detection via mTurquoise.
Parts Related to RecBCD
The RecBCD enzyme, also called Exonuclease V, is both a helicase that unwinds and separates linear DNA strands, and a nuclease that degrades linear DNA1. It consists of three subunits: RecB, RecC and RecD. RecBCD complex is essential for cell viability as it plays an important role in repair of DNA double-strand breaks by initiating homologous recombination2. However, RecBCD enzyme is also a highly processive at destruction of foreign DNA, like phage DNA3.RecBCD is regulated by a cis-acting DNA sequence known as Chi (Crossover Hotspot Instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity4. Since Chi binds RecBCD with a greater affinity than other dsDNA substrate, this can be used to inhibit RecBCD activity in TX-TL, to more efficiently express linear DNA in TX-TL.
We provide basic parts containing the full coding region of the subunit RecC (BBa_K2722000) and RecD (BBa_K2722001), which can be used for an independent study of these subunits. Moreover, you can find the corresponding inhibitor Chi6 (BBa_K2722002) in our parts collection.
Parts Related to mTurquoise
The fluorescent reporter protein mTurquoise can be used for a variety of applications. For example, it can be used to measure translation efficiency. Our parts collection includes the coding sequence of mTurquoise (BBa_K2722003) for easy fusion to target proteins or cloning into other functional plasmids, as well as an expression construct of mTurquoise with a constitutive promoter (BBa_K2722004).
The parts are flanked by a BioBrick prefix and suffix and contain pSB1C3 as a backbone and come from risk group 1 organisms.
To learn more about our parts visit the Registry.
iGEM Munich BioBrick List
<groupparts>iGEM18 Munich</groupparts>References
- Singleton, M. R., Dillingham, M. S., Gaudier, M., Kowalczykowski, S. C., & Wigley, D. B. (2004). Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature, 432(2014), 187
- Smith, G. R. (2012). How RecBCD enzyme and Chi promote DNA break repair and recombination: a molecular biologist's view. Microbiology and Molecular Biology Reviews, 76(2), 217-228.
- Smith, G. R. (2001). Homologous recombination near and far from DNA breaks: alternative roles and contrasting views. Annual review of genetics, 35(1), 243-274.
- Dillingham, M. S., & Kowalczykowski, S. C. (2008). RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiology and Molecular Biology Reviews, 72(4), 642-671.
