This construct was designed as a control for testing the effect of the Hin recombinase binding site hixC on transcription from the pBAD promoter.
Constructed from existing registry parts and parts created by the Davidson team.
Part is composed of a Hin recombinase expression casette driven by pLac and series of flippable units that include halves of GFP and RFP: T7prom-RBS-RFP1-hixC-GFP2-TT-hixC-RFP2-RBS-GFP1-hixC-RFP2-TT-hixC. The corresponding part without Hin does not glow. With Hin, most colonies do not glow, but green and red colonies appear. Evidence suggests that this is a result of Hin-mediated recombination.
Part is composed of a Hin recombinase expression casette driven by pLac and series of flippable units that include halves of GFP and RFP: T7prom-RBS-RFP1-hixC-RFP2-RBS-GFP1-hixC-RFP2-TT-hixC-GFP2-TT-hixC . The corresponding part without Hin glows redas a result of the expression of the hix-interrupted RFP. With Hin, most colonies are red, but green colonies appear. Evidence suggests that this is a result of Hin-mediated recombination.
This is the first computer that we will build. In its starting orientation there are no phenotypes expressed. There is the possibility of a false positive. There is only one true positive.
Alternatively, we may try building it in fewer steps doing a triple ligation.
Combine parts BBa_S03763 and BBa_S03764.
Construct for use in detecting the activity of Hin recombinase system. pBAD promoter is flanked by HixC recombination sites and may be flipped from the right to the left orientation. Tet resistance gene with upsteam RBS are also flanked by HixC sites and may be flipped from reverse to forward orientation. Cloned in pSB1A7 - no readthrough transcription detected.
Trascription readthrough into part appears to have been blocked by the introduction of double forward terminators before and after the cloning site.
This is an expression cassette for Tet resistence. The pBAD promoter is between two HixC sites, allowing for Hin recombinase to flip the pBAD promoter. This can turn on and off Tet resistence.
The pBAD is able to be flipped by the Hin recombinase.
Construct for use in detecting the activity of Hin recombinase system. pBAD promoter is flanked by HixC recombination sites and may be flipped from the right to the left orientation. Tet resistance gene with upsteam RBS are also flanked by HixC sites and may be flipped from forward to reverse orientation.
HixC is synthetic DNA, rest of construct is part J44003.
Should constitutively express the AraC protein (pBad inhibitor). HinLVA is under the control of pLac. Therefore, HinLVA should be expressed in the presence of IPTG.
The double forward terminator is placed uptream of pLac-HinLVA to prevent forward read-through transcription from the pSB1AK3 vector.
AraC-PC-TT - part BBa_S03591 pLac-HinLVA - part BBa_S03536
Construct for use in detecting the activity of Hin recombinase system. pBAD promoter is flanked by HixC recombination sites and may be flipped from the right to the left orientation. Tet resistance gene with upsteam RBS are also flanked by HixC sites and may be flipped from forward to reverse orientation. Cloned in pSB1A7 - readthrough transcription not detected.
This part contains the gene for tetracycline resistance flanked by HixC regions. This part will be used to test if the ribosome will be able to read through the Hix site.
This part as well as part BBa_J3103 are going to be used to find the optimal placement of the RBS.
(-1,-2) HixC-pBadrev-HixC : TetB-RBSrev-HixC
Part is composed of a Hin recombinase expression casette driven by pLac and series of flippable units that include halves of GFP and RFP: pLac-RBS-Hin-LVA-T7prom-RBS-RFP1-hixC-RFP2-RBS-GFP1-hixC-GFP2-TT-hixC-RFP2-TT-hixC. The corresponding part without Hin glows yellow as a result of the expression of the hix-interrupted GFP and RFP. With Hin, most colonies are yellow, but red and green colonies appear. Evidence suggests that this is a result of Hin-mediated recombination.
This part produces the Hin protein with an LVA degredation tag. We are currently looking into this as a way to ensure no Hin is floating around once we turn off the Hin promoter.
Salmonella typhimurium and the the Hin part without LVA.
AraC-PC (part BBa_I13458) should constitutively express the AraC protein (inhibitor of the pBad promoter). Expression of Hin invertase is under control of the pLac promoter. Therefore, Hin should be expressed in the presence of IPTG. This part was designed for two reasons: 1. To regulate DNA inversion (via controlled Hin invertase expression) on a second plasmid 2. To regulate the transcription of a pBad driven reporter (via the addition of arabinose) on the second plasmid
A double forward terminator is placed upstream of "pLac-Hin" to block forward read-through transcription from the pSB1AK3 vector. This should allow tighter control over Hin expression.
AraC-PC-TT - part BBa_S03591, a composite of BBa_I13458 and part BBa_B0015 pLac-RBS-Hin-TT - part BBa_S03537
Recombinational Enhancer (RE) for Hin/Hix inverting
This part contains the tetracycline resistance in the backwards orientation as well as a backwards RBS. These parts are flanked by HixC sites so when this part is placed downstream of the Hin or HinLVA cassettes the RBS and the tetracycline resistance will be in the correct orientation and the cell will express tetracyline resistance.
This part contains the coding region for tetracylcine resistance flanked by Hix C sites. This part is going to be used to test the optimal location of the RBS. If the Hin(BBa_J31000) or Hin LVA (BBa_J31001) genes are placed upstream of this part there is a possibility that the coding region between the Hix C sites is inverted.
This part is going to be used to test where the RBS needs to be placed.
This part contains the backwards coding region for the tetracycline resistance gene flanked by Hix C sites. If the Hin( BBaJ31000) ot the Hin-LVA (BBa_J31001) gene with a promoter and terminator, the backwards TetR gene will be inverted so that tetracyline resistance will be expressed.
DNA-invertase Hin from Salmonella typhimurium
Generates the Salmonella typhimurium Hin protein. This protein is used to invert DNA that is flanked by Hix siteds.
Construct for use in detecting the activity of Hin recombinase system. pBAD promoter is flanked by HixC recombination sites and may be flipped from the right to the left orientation. Tet resistance gene with upsteam RBS are also flanked by HixC sites and may be flipped from reverse to forward orientation.
HixC is synthetic, rest is from part BBa_S03626.
hixC binding site for Salmonella typhimurium Hin recombinase
Missouri Western and Davidson Groups, Todd Eckdahl
A 26 bp sequence of DNA composed of 12 bp inverted repeats and a 2 bp core that operates in Salmonella paired with a hixR binding site to recombine DNA. A second hix site is required for recombination to occur. The two sites bind Hin recombinase in the formation of an invertasome.
Standard BioBrick prefix and suffix were added to the 26 bp sequence.
Nanassy and Hughes. 1998. In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catlyzed by the Salmonella Hin Recombinase [http://www.genetics.org/cgi/content/abstract/149/4/1649]