Green fluorescent protein (E0240) is constitutively produced thanks to a constitutive promoter (J23119).
This part was build following the standard construction protocol.
J23119 is an E.coli constitutive promoter. The GFP sequence included in E0240 is a green fluorescent protein derived from jellyfish Aequeora victoria wild type GFP.
a plasmid with EGFP reporter to report double repressed promoter activity
EGFP with strong SD signal, a MCS before EGFP to insert double repressed promoter, high efficient double terminator T1T2 locates on both upstream of MCS and downstream of EGFP.
This part contains a green fluorescence protein (GFP) under control of a CMV promoter and its double terminator (BBa_I712669 + BBa_B0014). This part is reliable to use when we want to know if a gene is inserted into a eukaryotic cell. For example we can use it when we design a recombinant virus. In fact we inserted this bio-brick into the synthetic virus; finally we are able to see if the virus can infect the targeted cells. This bio-brick already contains a terminator, so it is ready to use.
Part:BBa_I712669, Designed by Rok Gaber Group: iGEM07_Ljubljana (2007-10-24) Part:BBa_B0014, Designed by Reshma Shetty Group: Registry (2003-07-16)
Platform for terminators analysis under the control of T7 promoter
We used this part to test terminators: the two subsequent proteins allow to quantify the termination efficiency as the ratio between Venus's and Cherry's fluorescence. The vector is pET21b, that contains lacI, t7 promoter and ampicillin resistance gene. The two fluorescent proteins have partially overlapping emission spectra, thus the fluorimetric measures should be taken with these parameters, to avoid interference between the two: Venus excitation: 485nm, Venus emission: 528nm Cherry excitation: 528nm, Cherry emission: 615nm We inserted the prefix-suffix linker between the two proteins, to allow terminator characterization.
Using and modifying this construct is important to remember that the two proteins have strong N-terminal homology, especially designing primers. For any future improvement, maybe it would be better two use two different fluorescence proteins, because these two has partially overlapping emission spectra and different folding kinetics. Using and modifying this construct is important to remember that the two proteins have strong N-terminal homology, especially designing primers. For any future improvement, maybe it would be better two use two different fluorescence proteins, because these two has partially overlapping emission spectra and different folding kinetics.
It's made from a Roberta Lentini's construct (from Mansy lab) that consist of pET21b with the two proteins and a 20bp linker between them. That construct was mutated two times to eliminate illegal restriction sites; prefix-suffix linker was added by insertion PCR.
Low to medium copy BioBrick standard vector
pSB3K3-1 is a medium copy plasmid with kanamycin resistance. It has a p15A pMR101-derived replication origin, copy number 20-30. (Lutz and Bujard, 1997) pSB3K3-1 has a terminator upstream of its MCS, which is oriented to prevent transcription from *inside* the MCS from reading out into the vectorl. A second terminator (E.coli His operon-derived) is downstream of the MCS, again insulating the vector from transcription reading out of the MCS. Ideally, future versions of standard biobrick vectors would have terminators bracketing the MCS that were 100% efficient in terminating transcription both into and out of the MCS region.