Difference between revisions of "Part:BBa K2539500"

Revision as of 05:54, 11 October 2018

PalcA-Regulated GFP Expression Construct

This construct combines two of our submitted composite parts: a constitutively expressed alcR construct (BBa_K2539300) and a PalcA-regulated GFP expression construct (BBa_K2539500). PalcA is an inducible promoter (BBa_K2092002), which can be activated in the presence of both ethanol and a transcription factor, alcR (BBa_K2092001). It was originally isolated from the fungus Aspergillus nidulans (Panozzo et al., 1997). AlcR is a gene found in Aspergillus nidulans which encodes for a regulatory protein (Panozzo et al., 1997). AlcR can bind to the alcA and aldA promoters (PalcA and PaldA, respectively). In the presence both AlcR and ethanol or threonine, genes downstream of these promoters are expressed (Felenbok et al., 1988). GFP is used as a visible reporter so we can characterize the function of the promoter PalcA.

Construct Design

This construct constitutively express alcR, a regulatory protein necessary for the activation of PalcA. We use a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the alcR sequence (BBa_K2092001), and a double terminator (BBa_B0015) to end transcription. Behind this, the PalcA promoter is placed in front of a strong RBS (BBa_B0034), GFP (BBa_E0040), and a double terminator (BBa_B0015). Together, this construct expresses alcR, which can activate the PalcA promoter and the expression of GFP if ethanol is present.

PCR Check Results

The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.

PCR check for BBa_K2539550 using VF2 and VR primers. Using these primers, PCR produced a band at the expected size of 4.8 kb.

Characterization

References

Felenbok B, Sequeval D, Mathieu M, Sibley S, Gwynne DI, Davies RW. (1988). The ethanol regulon in Aspergillus nidulans: characterization and sequence of the positive regulatory gene alcR. Gene, 73(2), 385–396.

Panozzo C, Capuano V, Fillinger S, Felenbok B. (1997). The zinc binuclear cluster activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. J Biol Chem. 5;272(36):22859-65.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 274
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1513

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2539500 short</partinfo>
-This construct uses the inducible promoter PalcA (BBa_K2092002). PalcA is activated in the presence of both ethanol and a transcription factor, alcR (BBa_K2092001), and was originally isolated from the fungus <i>Aspergillus nidulans</i> (Panozzo <i>et al.</i>, 1997). GFP is used as a visible reporter so we can characterize the function of the promoter PalcA.
+This construct combines two of our submitted composite parts: a constitutively expressed alcR construct (BBa_K2539300) and a PalcA-regulated GFP expression construct (BBa_K2539500).
+PalcA is an inducible promoter (BBa_K2092002), which can be activated in the presence of both ethanol and a transcription factor, alcR (BBa_K2092001). It was originally isolated from the fungus <i>Aspergillus nidulans</i> (Panozzo <i>et al.</i>, 1997). AlcR is a gene found in <i>Aspergillus nidulans</i> which encodes for a regulatory protein (Panozzo <i>et al.</i>, 1997). AlcR can bind to the alcA and aldA promoters (PalcA and PaldA, respectively). In the presence both AlcR and ethanol or threonine, genes downstream of these promoters are expressed (Felenbok <i>et al.,</i> 1988).
+GFP is used as a visible reporter so we can characterize the function of the promoter PalcA.
@@ Line 9: / Line 11: @@
 <b><font size="+1">Construct Design</font></b>
-https://static.igem.org/mediawiki/parts/7/74/T--TAS_Taipei--500construct.jpg
+https://static.igem.org/mediawiki/parts/4/4c/T--TAS_Taipei--550construct.jpg
-The PalcA promoter is placed in front of a strong RBS (BBa_B0034), GFP (BBa_E0040), and double terminator (BBa_B0015). This places the expression of GFP under the control of the PalcA promoter.
+This construct constitutively express alcR, a regulatory protein necessary for the activation of PalcA. We use a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the alcR sequence (BBa_K2092001), and a double terminator (BBa_B0015) to end transcription. Behind this, the PalcA promoter is placed in front of a strong RBS (BBa_B0034), GFP (BBa_E0040), and a double terminator (BBa_B0015).
+Together, this construct expresses alcR, which can activate the PalcA promoter and the expression of GFP if ethanol is present.
@@ Line 21: / Line 24: @@
 https://static.igem.org/mediawiki/parts/a/a2/T--TAS_Taipei--300pcr.jpg
-<b>PCR check for BBa_K2539500 using VF2 and VR primers. Using these primers, PCR produced a band at the expected size of 2 kb.</b>
+<b>PCR check for BBa_K2539550 using VF2 and VR primers.</b> Using these primers, PCR produced a band at the expected size of 4.8 kb.
+<b><font size="+1">Characterization</font></b>
 <b><font size="+1">References</font></b>
+Felenbok B, Sequeval D, Mathieu M, Sibley S, Gwynne DI, Davies RW. (1988). The ethanol regulon in Aspergillus nidulans: characterization and sequence of the positive regulatory gene alcR. Gene, 73(2), 385–396.
 Panozzo C, Capuano V, Fillinger S, Felenbok B. (1997). The zinc binuclear cluster activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. J Biol Chem. 5;272(36):22859-65.