Difference between revisions of "Part:BBa K642004"

Latest revision as of 23:58, 28 September 2011

TetR repressor tagged with yBFP and a VP16 activation domain

This is the repressor TetR from the Tn10 tetracycline (Tc) resistance operon of E.coli. It will bind with high specificity to the tetO operator and is inhibited in the presence of ATc (1). This repressor is tagged with yeast codon optimized BFP as well as a VP16 activation domain. BFP is a monomeric fluorescent protein that has an excitation peak of 399 nm and an emission peak of 465 nm (2). It was yeast codon optimized through DNA synthesis for the purpose of expressing in S. cerevisiae tagged to various repressors and activators. VP16 is a transcription activation domain from the herpes simplex virus that will initiate transcription when fused to a repressor protein. (3)

Fluorescence Data

We quantified the fluorescence of two tagged repressors: BBa_K642000 and BBa_K642004 by cloning them downstream of the yeast Gal10/1 promoter. Each construct was then integrated using KanMX6 selection into the Ade2 locus of a BY4742 strain of S.cerevisiae. The resulting strain was innoculated in 3 mL of 1x SM + 2% Galactose + 2% Adenine overnight. The overnight culture was reinoculated into 1 mL of the same media at an OD600 of 0.02. After three hours the strain was analyzed for BFP fluorescence on a Cyan ADP flow cytometer.

Figure 1: In vivo BFP fluorescence of BBa_K642000, a TetR repressor tagged with yeast codon optimized BFP and BBa_K642004, a TetR repressor tagged with yeast codon optimized BFP and VP16 activation domain. These parts were integrated using KanMX6 selection into a BY4742 strain of S. cerevisiae.

References

(1) Hillen, W. & Berens, C. (1994). "Mechanisms underlying expression of Tn10 encoded tetracycline resistance." Annu Rev Microbiol 48:345-69.

(2) Subach, O. M., I. S. Gundorov, et al. (2008). "Conversion of red fluorescent protein into a bright blue probe." Chem Biol 15(10): 1116-24.

(3) Urlinger et al. (2000). "Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity." Proc Natl Acad Sci U S A 97(14):7963-8.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K642004 short</partinfo>
-This is the repressor TetR from the Tn10 tetracycline (Tc) resistance operon of E.coli. It will bind with high specificity to the tetO operator and is inhibited in the presence of ATc (1). This repressor is tagged with yeast codon optimized BFP as well as a VP16 activation domain. BFP is a monomeric fluorescent protein and has an excitation peak of 399 nm and an emission peak of 465 nm (2). It was yeast codon optimized through DNA synthesis for the purpose of expressing in S. cerevisiae tagged to various repressors and activators.
+This is the repressor TetR from the Tn10 tetracycline (Tc) resistance operon of ''E.coli''. It will bind with high specificity to the ''tetO'' operator and is inhibited in the presence of ATc (1). This repressor is tagged with yeast codon optimized BFP as well as a VP16 activation domain. BFP is a monomeric fluorescent protein that has an excitation peak of 399 nm and an emission peak of 465 nm (2). It was yeast codon optimized through DNA synthesis for the purpose of expressing in ''S. cerevisiae'' tagged to various repressors and activators. VP16 is a transcription activation domain from the herpes simplex virus that will initiate transcription when fused to a repressor protein. (3)
+===Fluorescence Data===
+We quantified the fluorescence of two tagged repressors: BBa_K642000 and BBa_K642004 by cloning them downstream of the yeast Gal10/1 promoter. Each construct was then integrated using KanMX6 selection into the Ade2 locus of a BY4742 strain of ''S.cerevisiae''. The resulting strain was innoculated in 3 mL of 1x SM + 2% Galactose + 2% Adenine overnight. The overnight culture was reinoculated into 1 mL of the same media at an OD600 of 0.02. After three hours the strain was analyzed for BFP fluorescence on a Cyan ADP flow cytometer.
+[[Image:BFP fluorescence for tagged TFs.png]]
+Figure 1: In vivo BFP fluorescence of BBa_K642000, a TetR repressor tagged with yeast codon optimized BFP and BBa_K642004, a TetR repressor tagged with yeast codon optimized BFP and VP16 activation domain. These parts were integrated using KanMX6 selection into a BY4742 strain of ''S. cerevisiae''.
 ===References===