Difference between revisions of "Part:BBa K208000"

Latest revision as of 15:04, 22 October 2009

GFP
This is a new Silver-fusion compatible GFP BioBrick part. This particular GFP was previously mutated for improved fluorescence photostability (Crameri, 1996). The excitation and emission wavelengths for this GFP are 395 nm and 509 nm, respectively. That being said, GFP-positive cells emit a bright green fluorescence when exposed to shorter-wavelength UV light, such as on a transilluminator. See part BBa_K208045 for a functional composite part containing this GFP with a Lac promoter.

Usage and Biology

Many mutant forms of GFP have been created which improve fluorescence photostability and ultimately the ability of GFP to function as a practical reporter. The cycle 3 mutant developed by Crameri et al. (1996) is of special interest because it produces a fluorescence signal 45-fold greater than wild-type GFP. The developed GFP possesses three point mutations of the wild-type GFP. These mutations do not affect the chromophore itself, but reside in the surrounding barrel of the GFP protein. In E. coli, due to its hydrophobic nature, most of the wild-type GFP gathers to form inclusion bodies that limit the ability of blue light to provide the necessary excitation energy to activate fluorescence (Crameri , 1996). The three point mutations in the cycle 3 mutant, have no effect on excitation and emissions maxima, but create a more hydrophilic GFP less prone to form inclusion bodies. The soluble mutant is easily activated by a UV light box or light wand common in the laboratory creating an immediate, practical reporter protein. Furthermore, fusions onto amino- or carboxy-termini of GFP do not inhibit fluorescence, which makes GFP an ideal candidate for fusion studies (LaVallie, 1995).

The Silver-fusion compatibility means that the prefix and suffix are slightly modified from the original BioBrick prefix and suffix. Specifically, a single base pair is removed from the prefix and suffix, directly next to the part.

The Silver prefix is: 5' GAATTC GCGGCCGC T TCTAGA 3'
The Silver suffix is: 5' ACTAGT A GCGGCCG CTGCAG 3'

The scar that is formed from ligating XbaI and SpeI between two fusion compatible parts is 5' ACTAGA 3', which is six base pairs long and does not contain a stop codon. Therefore, fusion of two proteins can occur by this method. Additionally, this part is still useful and functional for non-fusion applications. There are more design considerations for when using Silver BioBrick parts. See this description by the Silver lab for more information about Silver fusion: http://www.openwetware.org/wiki/Silver:_BB_Strategy

Sequence and Features