Team:Edinburgh/GenBrick/GenbrickAction

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Revision as of 17:54, 4 October 2013

Introduction
Genbrick in action
Linker designer
Appendix

Furbox GenBrick Assembly

Using the protocol found in the appendix, a 3-part construct was assembled with an acceptor vector pSB1C3 (BBa_K1122009), a Lac promoter and truncated gene called PLac_LacZ (BBa_J33207) optimised for GenBrick and a fluorescent protein, GFP (BBa_K1122004) (Figure 1). The aim was to test an inverted repeat sequence known as the fur box. When internal concentrations of iron are high within a cell, the Fur protein binds to the fur box and represses the gene upstream from it. The consensus sequence for the fur box is ‘TGATAATAATTATCA’. The eye and hook linkers between PLac_LacZ and GFP were engineered to have a fur box, an RBS and a start codon.

Spacer with STOP-Fur box-RBS-START

.........................STOP.......................Fur-box..................RBS..........................START...............

5’GCCCCAAGTTAATCATGATAATAATTATCAAGGAGGTGGAGCGGTATGGGTAGT...3’

Hook Long 6: 5’GCCCCAAGTTAATCATGATAATAATTATCAAGGA3’

Hook Short 6: 5’ATCATGATTAACTTGG3’

Eye Long 6: 5’CGAACTACCCATACCGCTCCACCTCCTTGATAATTATT3’

Eye Short 6: 5’GGTGGAGCGGTATGGGTAGT3’

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 {{Team:Edinburgh/Genabler_side_menu}}
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+==='''Furbox GenBrick Assembly'''===
+Using the protocol found in the appendix, a 3-part construct was assembled with an acceptor vector pSB1C3 (BBa_K1122009), a Lac promoter and truncated gene called PLac_LacZ (BBa_J33207) optimised for GenBrick and a fluorescent protein, GFP (BBa_K1122004) (Figure 1). The aim was to test an inverted repeat sequence known as the fur box. When internal concentrations of iron are high within a cell, the Fur protein binds to the fur box and represses the gene upstream from it. The consensus sequence for the fur box is ‘TGATAATAATTATCA’. The eye and hook linkers between PLac_LacZ and GFP were engineered to have a fur box, an RBS and a start codon.
+'''Spacer with STOP-Fur box-RBS-START'''
+<html>
+.........................STOP.......................Fur-box..................RBS..........................START...............
+</html>
+’<span style="color: red;">GCC</span><span style="color: green;">CCAAGT<span style=”background-color: #D8D8D8”>TAA</span>TCA<span style=”background-color: yellow”>TGATAATAATTATCA</span><span style=”background-color: grey”>AGGA</span></span><span style="color: orange;"><span style=”background-color: grey”>GGT</span>GGAGCGGT<span style=”background-color: grey”>ATG</span>GGTAGT</span>...3’
+Hook Long 6: 5’GCCCCAAGTTAATCATGATAATAATTATCAAGGA3’
+Hook Short 6: 5’ATCATGATTAACTTGG3’
+Eye Long 6: 5’CGAACTACCCATACCGCTCCACCTCCTTGATAATTATT3’
+Eye Short 6: 5’GGTGGAGCGGTATGGGTAGT3’