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From 2010.igem.org

Used BioBricks

This is a list of all BioBricks we used from the registry or created by ourselves and a short explanation, why we used them. The list is sorted alphabetically.

BioBrick (name and partnumber)	Status	Comment
double terminator, <partinfo>B0017</partinfo>	from registry	this contains two <partinfo>B0010</partinfo> terminators we used this because of problems mentioned in registry with <partinfo>B0012</partinfo>
RBS, <partinfo>B0034</partinfo>	from registry	we always assembled a strong RBS
lacI gene, <partinfo>C0012</partinfo>	from registry	used this to build the tightly regulated lac-operon <partinfo>K389050</partinfo>
mRFP, <partinfo>E1010</partinfo>	from registry	this BioBrick was used as a reporter gene, e.g. in the construct <partinfo>K389013</partinfo>
lacIq promoter, <partinfo>I14032</partinfo>	from registry	used this to build the tightly regulated lac-operon <partinfo>K389050</partinfo>
mRFP generator, <partinfo>J04450</partinfo>	from registry	this BioBrick was used as a visible selection marker for cloning other BioBricks into the <partinfo>pSB1C3</partinfo> plasmid
constitutive promoter, <partinfo>J23110</partinfo>	from registry	we always used a medium strong constitutive promoter from the registry to express our BioBricks this BioBrick was used e.g. in <partinfo>K389010</partinfo> or <partinfo>K389011</partinfo>
R6K origin, <partinfo>J61001</partinfo>	from registry	this BioBrick was used to create a plasmid without ColE1 ori the R6K ori only works in some E. coli strains, so it is used for a two plasmid screening system
virA, <partinfo>K238008</partinfo>	from registry, fixed	we wanted to use the virA BioBrick from the registry but problems occurred, so we created our own virA from Agrobacterium tumefaciens C58 TI-plasmid our own (working) virA is here: <partinfo>K389001</partinfo>
vir-promoter, <partinfo>K238011</partinfo>	from registry, fixed	same as the virA BioBrick - this one from the registry does not work properly, so we made a vir-promoter by ourselves the part that was sent to us is actually a tetR gene (<partinfo>C0040</partinfo>) under the control of a constitutive promoter (<partinfo>J23105</partinfo>) before a double terminator (<partinfo>B0015</partinfo>, compare our sequencing results)
virA, <partinfo>K389001</partinfo>	new	"our" virA illegal PstI restriction site removed by site-directed mutagenesis isolated from A. tumefaciens C58 TI-plasmid
virG, <partinfo>K389002</partinfo>	synthesized, new	virG that works in E. coli without rpoA gene from A. tumefaciens mutations ... + optimized codon-usage (E. coli) + removal of all illegal restriction sites because it was synthesized
vir-promoter, <partinfo>K389003</partinfo>	new	"our" vir-promoter is induced by phosphorylated VirG isolated from A. tumefaciens C58 TI-plasmid
firefly luciferase, <partinfo>K389004</partinfo>	new	"our" luciferase sensitive reporter gene isolated from Promega's pGL4.10luc2 plasmid
Kanamycin resistance gene, <partinfo>K389005</partinfo>	new	"our" antibiotic resistance used for directed evolution of virA and mutated virA screenings, respectively isolated from the BioBrick <partinfo>P1003</partinfo>
virA generator, <partinfo>K389010</partinfo>	composite	medium strong constitutive promoter, so there is enough VirA receptor expressed but not too much so the cell suffers from the expression ...
virA screening device, <partinfo>K389011</partinfo>	composite	the new virG which works without rpoA is expressed constitutively and a kanamycin resistance is gene under the control of a vir promoter to screen mutants of virA (which are on a different plasmid in a <partinfo>K389010</partinfo>-like part) the better the VirA receptor recognizes a substance the more resistant the cell is against kanamyin this device has to be on a plasmid with different ori than the virA which should be screened (different compatibility classes) we cloned this BioBrick into a plasmid with R6K ori which only works in pir+ or pir116 strains (e.g. E. coli EC100D), so we can easily seperate the two plasmids by transforming them into strains without Pir protein (e.g. E. coli TOP10)
virA reporter device with luc, <partinfo>K389012</partinfo>	composite	this BioBrick is similar to <partinfo>K389011</partinfo> but with the reporter luciferase instead of an antibiotic resistance gene with this BioBrick it is possible to measure the activity of the vir promoter and the VirA receptor, respectively
virA reporter device with mRFP, <partinfo>K389013</partinfo>	composite	this BioBrick is similar to <partinfo>K389011</partinfo> but with the reporter mRFP instead of an antibiotic resistance gene with this BioBrick it is possible to measure the activity of the vir promoter and the VirA receptor, respectively
Test, <partinfo>K389014</partinfo>	composite	this BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo> used for testing the general screening concept of expressing an antibiotic resistance with a vir promoter after induction with acetosyringone (the natural VirA inductor)
vir promoter characterisation part with luc, <partinfo>K389015</partinfo>	composite	...
vir promoter characterisation part with mRFP, <partinfo>K389016</partinfo>	composite	...
vir promoter part, <partinfo>K389017</partinfo>	composite	...
tightly controlled lac operator, <partinfo>K389050</partinfo>	composite	...
Kanamycin-resistance cassette, <partinfo>P1003</partinfo>	from registry	we used this BioBrick to create <partinfo>K389005</partinfo>
ccdB-gene, <partinfo>P1010</partinfo>	from registry	we used this BioBrick for 3A-assemblies
high-copy plasmid with Amp and Tet resistance, <partinfo>pSB1AT3</partinfo>	from registry	we used this plasmids in 3A-assemblies and as the backbone of the mutated and unmutated <partinfo>K389010</partinfo> part
high-copy plasmid with Cm resistance, <partinfo>pSB1C3</partinfo>	from registry	the plasmid to send in the BioBricks
lac operator, <partinfo>R0010</partinfo>	from registry	used to build a tightly regulated lac operator (<partinfo>K389050</partinfo>)