Difference between revisions of "Part:BBa K323088"

Latest revision as of 13:44, 27 October 2010

In vivo testing device for protein-DNA binding: part 1 (DTER_pSYN_BbsI)

Device for in vivo testing of protein-DNA binding.

To test binding of DNA binding proteins to a corresponding specific target DNA sequence in vivo we designed a device composed of several parts:

1. this part is a synthetic promoter pSYN in which a DNA binding sequence, particular for each DNA-binding protein to be tested, could be inserted between -35 and -10 sites using BbsI restriction site;

2. lacZ reporter gene (Part:BBa_I732019), which expression is controlled by pSYN;

3. DNA binding protein (cut with XbaI/NotI) to be tested under arabinose inducible (pBAD) promoter in lacZ_DTER_pBAD_BsaI_DTER cut with BsaI (Part:BBa_K323089).

The successful binding of DNA binding protein to the synthetic promoter would prevent transcription of lacZ resulting in lower beta-galactosidase activity.

Figure: BbsI restriction site. The BbsI restriction endonuclease cuts the DNA outside of its recognition site. Primers for the annealing of the operator sequences were designed to fit into the clone in site, that is GACA sequence at the 5` end of the forward primer and AATA sequence at the 3` end of the reverse primer.

Figure: Device for in vivo testing of protein-DNA binding. A) Expression of beta-galactosidase with no arabinose present: Expression of the DNA binding protein is regulated by the pBAD promoter. With no arabinose to induce the promoter, the DNA binding protein cannot be transcribed and therefore cannot bind to its operator sequence, inserted into the pSYN promoter. The lacZ gene is thus transcribed and the measured beta-galactosidase activity high. B) Expression of beta-galactosidase with arabinose present: With arabinose added to the media, the pBAD promoter is induced, the DNA binding protein is transcribed and bound to its operator sequence in the pSYN promoter. Therefore the promoter is inactive and the lacZ gene cannot be transcribed, which results in a low beta-galactosidase activity.

The 2010 iGEM team Slovenia have tested seven DNA binding proteins (zinc fingers HivC, Gli1, Zif268, Jazz, Blues, PBSII and the TAL transcription factor) with this device. For results of the experiment see tab Experience.

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_K323088 short</partinfo>
-This part contains a double terminator ([[Part:BBa_B0015]]), and a synthetic promoter (pSYN), designed to contain a ''Bbs''I restriction site (operator clone in site). The terminator in front of the promoter serves the purpose of stopping the translation under promoters present in the vector. Combined with [[Part:BBa_K323089]] (containing the corresponding DNA binding protein), this part forms a '''universal testing device for DNA binding proteins'''.
-[[Image:PSYN_Bbs.png|center|thumb|800px| '''Figure: ''Bbs''I restriction site.''' The ''Bbs''I restriction endonuclease cuts the DNA outside of its recognition site. Primers for the annealing of the operator sequences were designed to fit into the clone in site, that is TTAT sequence at the 3` end of the forward primer and CTGT sequence at the 5` end of the reverse primer.]]
+'''Device for ''in vivo'' testing of protein-DNA binding'''.
+To test binding of DNA binding proteins to a corresponding specific target DNA sequence ''in vivo'' we designed a device composed of several parts:
+. this part is a synthetic promoter pSYN in which a DNA binding sequence, particular for each DNA-binding protein to be tested, could be inserted between -35 and -10 sites using ''Bbs''I restriction site;
+. ''lac''Z reporter gene ([[Part:BBa_I732019]]), which expression is controlled by pSYN;
+. DNA binding protein (cut with ''Xba''I/''Not''I) to be tested under arabinose inducible (pBAD) promoter in lacZ_DTER_pBAD_BsaI_DTER cut with BsaI ([[Part:BBa_K323089]]).
+The successful binding of DNA binding protein to the synthetic promoter would prevent transcription of lacZ resulting in lower beta-galactosidase activity.
+[[Image:PSYN_Bbs.png|center|thumb|500px| '''Figure: ''Bbs''I restriction site.''' The ''Bbs''I restriction endonuclease cuts the DNA outside of its recognition site. Primers for the annealing of the operator sequences were designed to fit into the clone in site, that is GACA sequence at the 5` end of the forward primer and AATA sequence at the 3` end of the reverse primer.]]
+[[Image:Univ._sistem_princip.png|center|thumb|500px|'''Figure: Device for in vivo testing of protein-DNA binding.''' '''A) Expression of beta-galactosidase with no arabinose present:''' Expression of the DNA binding protein is regulated by the pBAD promoter. With no arabinose to induce the promoter, the DNA binding protein cannot be transcribed and therefore cannot bind to its operator sequence, inserted into the pSYN promoter. The ''lac''Z gene is thus transcribed and the measured beta-galactosidase activity high. '''B) Expression of beta-galactosidase with arabinose present:''' With arabinose added to the media, the pBAD promoter is induced, the DNA binding protein is transcribed and bound to its operator sequence in the pSYN promoter. Therefore the promoter is inactive and the ''lac''Z gene cannot be transcribed, which results in a low beta-galactosidase activity.]]
+The 2010 iGEM team Slovenia have tested seven DNA binding proteins (zinc fingers HivC, Gli1, Zif268, Jazz, Blues, PBSII and the TAL transcription factor) with this device. For results of the experiment see tab [https://parts.igem.org/Part:BBa_K323088:Experience Experience].
-[[Image:Univ._sistem_princip.png|left|thumb|600px|'''Figure: Universal testing device for DNA binding proteins.''' '''A) Expression of beta-galactosidase with no arabinose present:''' Expression of the DNA binding protein is regulated by the pBAD promoter. With no arabinose to induce the promoter added to the media, the DNA binding protein cannot be transcribed and therefore cannot bind to its operator sequence, inserted into the pSYN promoter. The ''lac''Z gene is thus transcribed and the measured beta-galactosidase activity high. '''B) Expression of beta-galactosidase with arabinose present:''' With arabinose added to the media, the pBAD promoter is induced, the DNA binding protein is transcribed and bound to its operator sequence in the pSYN promoter. Therefore the promoter is inactive and the ''lac''Z gene cannot be transcribed, which results in a low beta-galactosidase activity.]]
-Binding can be tested with the beta-galactosidase assay (for detailed description of the method see the 2010 iGEM team Slovenia wiki). The activity of a beta-galactosidase enzyme, expressed under the promotor with a binding site for a DNA binding protein, is measured. By this principle the binding strength of a chosen DNA binding protein to its binding sequence can be determined - the stronger the binding, the lower the expression of beta-galactosidase under the pSYN promoter.
-The 2010 iGEM team Slovenia have tested seven DNA binding proteins (zinc fingers HivC, Gli1, Zif268, Jazz, Blues, PBSII and the TAL transcription factor) with this device. For results of the experiment see tab "Experience".