Team:Bielefeld-Germany/Results/Used

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Used BioBricks

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

BioBrick (name and partnumber) Status Comment
double terminator, BBa_B0017:

B0010

B0010
from registry

This is a double terminator which contains two BBa_B0010 terminators. We used this double terminator instead of the commonly used BBa_B0015 double terminator (which was assembled from the BBa_B0010 and the BBa_B0012 terminator) because of problems mentioned in the partsregistry with the BBa_B0012 terminator.

RBS, BBa_B0034:

B0034
from registry

The strong ribosomal binding site (RBS) AGGAG was always assembled.

lacI gene, BBa_C0012:
lacI
C0012
from registry

This is a gene for the lac repressor lacI with a LVA degradation tag. We used this BioBrick to build a tightly regulated lac-operon BBa_K389050 with low basal transcription. It seems that this lacI is either degraded too quickly or it does not work properly because it is not possible to repress the lac operon with this part.

mRFP, BBa_E1010:
mRFP1
E1010
from registry

This BioBrick was used as a reporter gene, e.g. in the construct BBa_K389013. mRFP as a reporter gene has the advantage that it is easy to measure and comparatively stable.

lacIq promoter, BBa_I14032:
P(Lac) IQ
I14032
from registry

This promoter is a strong constitutive promoter which is natively located upstream of the lacI gene in E. coli lacIq strains. It was used to build a tightly regulated lac-operon BBa_K389050 in which it was assembled upstream of the lacI gene from the partsregistry BBa_C0012.

sensitivity tuner, BBa_I746370:

I746313

I746320
from registry

We used this BioBrick to amplify our read-out signal luciferase in the part BBa_K389401. This BioBrick is supposed to amplify the effect of a PoPS device 15 - 20 fold compared to a system without sensitivity tuner (Cambridge, iGEM 2007, amplifier project).

sensitivity tuner, BBa_I746380:

I746314

I746320
from registry

We used this BioBrick to amplify our read-out signal luciferase in the part BBa_K389402. This BioBrick is supposed to amplify the effect of a PoPS device 10 fold compared to a system without sensitivity tuner (Cambridge, iGEM 2007, amplifier project).

sensitivity tuner, BBa_I746390:

I746315

I746320
from registry

We used this BioBrick to amplify our read-out signal luciferase in the part BBa_K389403. This BioBrick is supposed to amplify the effect of a PoPS device 30 - 35 fold compared to a system without sensitivity tuner (Cambridge, iGEM 2007, amplifier project).

mRFP generator, BBa_J04450:
LacI
R0010

B0034
mRFP1
E1010

B0015
from registry

This BioBrick was used as a visible selection marker for cloning other BioBricks into the pSB1C3 plasmid to submit them to the registry. The promoter in this mRFP generator is pretty strong so it is easy to separate colonies which carry this BioBrick from colonies which do not carry this BioBrick anymore.

strong constitutive promoter, BBa_J23102:

J23102
from registry

A strong constitutive promoter which was used to create BioBrick BBa_K389318 to test the firefly luciferase BioBrick BBa_K389004 and the luciferase measurement system.

weak constitutive promoter, BBa_J23103:

J23103
from registry

A weak constitutive promoter which was used to create BioBrick BBa_K389302 to test the firefly luciferase BioBrick BBa_K389004 and the luciferase measurement system.

medium strong constitutive promoter, BBa_J23110:

J23110
from registry

This medium strong constitutive promoter from the registry was assembled before our BioBricks, so they are neither expressed too strong, so the cell suffers from metabolomic stress, nor too weak, so there is no visible effect of our BioBricks. This BioBrick was used e.g. in BBa_K389010 or BBa_K389011.

medium strong constitutive promoter, BBa_J23115:

J23115
from registry

A medium strong constitutive promoter which was used to create BioBrick BBa_K389307 to test the firefly luciferase BioBrick BBa_K389004 and the luciferase measurement system.

R6K origin, BBa_J61001:
R6K
J61001
from registry

This BioBrick was used to create a plasmid without ColE1 ori. The R6K ori has a different compatibility class than the ColE1 ori. So it is used for a two plasmid screening system. The R6K ori is a medium copy plasmid in pir+ / pir116 E. coli strains like EC100D but it does not replicate in pir- strains like E. coli TOP10. So it is easy to separate a ColE1 ori plasmid from another with R6K ori by simply transforming both to e.g. E. coli TOP10. The R6K ori was cloned into the pSB1C3 plasmid. Subsequently, the plasmid containing the R6K ori was digested with Hin6I. The largest fragment was extracted from an agarose gel and ligated with T4 DNA Polymerase. Thereby the ColE1 ori was removed, leaving a small fragment of about 30 bp.

virA, BBa_K238008:
VirA
K238008
from registry, fixed

We wanted to use this virA BioBrick from the registry but problems occurred ( characterization), so we created our own virA from Agrobacterium tumefaciens C58 TI-plasmid. Our own (working) virA has the partnumber BBa_K389001.

vir-promoter, BBa_K238011:
VirB
K238011
from registry, fixed

The same problems occurred as with the virA BioBrick - this one from the registry does not work properly, so we made a vir-promoter by ourselves (BBa_K389003). The part that was sent to us is actually a tetR gene (BBa_C0040) under the control of a constitutive promoter (BBa_J23105) before a double terminator (BBa_B0015, compare our sequencing results).

virA, BBa_K389001:
virA
K389001
new

The VirA receptor is used by A. tumefaciens to detect acetosyringone and other phenolic substances which are secreted by plants after injury. In presence of these substances VirA phosphorylates itself and afterwards VirG, a response regulator which activates vir promoters. These promoters control genes which are used for infecting the injured plant. This virA gene was isolated from the TI-plasmid of A. tumefaciens C58. An illegal PstI restriction site was removed by site-directed mutagenesis. The functionality of this BioBrick was proved with BBa_K389015 and BBa_K389016.

mutated virG, BBa_K389002:
virG
K389002
synthesized, new

This is the VirG response regulator from the VirA/G receptor system. The VirA/G receptor system is used by A. tumefaciens to detect phenolic substances secreted by injured plants, e.g. acetosyringone. In presence of these substances, VirG is activated by the VirA receptor and induces the transcription of genes under the control of a vir promoter. Normally, VirG needs the rpoA gene (RNA polymerase subunit) from A. tumefaciens to work in Escherichia coli but this virG BioBrick is mutated, so it works with the rpoA subunit from E. coli. For this reason the point mutations G56V and I77V were brought into the gene (YC Jung et al., 2004). Because this BioBrick is synthesized (Mr. Gene GmbH), codon usage is optimized for E. coli and illegal restriction sites were removed. When you use this virG gene in a VirA/G signaling system you do not need BBa_K238010 anymore to get the system working in E. coli.

vir-promoter, BBa_K389003:
Pvir
K389003
new

Vir-promoters from A. tumefaciens are induced by phosphorylated VirG response regulators and control genes for infecting plants in their natural host. They are part of the VirA/G signal transduction system. This is a vir promoter from A. tumefaciens C58. It is located on the TI-plasmid upstream the virB genes.

firefly luciferase, BBa_K389004:
luc
K389004
new

This is a BioBrick for a firefly (Photinus pyralis) luciferase. It is a sensitive reporter gene. It was isolated from Promega's pGL4.10[luc2] plasmid.

Kanamycin resistance gene, BBa_K389005:
kanR
K389005
new / modified

This is a kanamycin / neomycin resistance gene without promoter or RBS. It is used for directed evolution of virA and mutated virA screenings, respectively. It was isolated from the BioBrick BBa_P1003 (a kanamycin resistance gene with promoter and RBS).

virA generator, BBa_K389010:

J23110

B0034
virA
K389001
composite

This part is for expressing a VirA receptor. A medium strong constitutive promoter is used, so there is enough VirA receptor expressed but not too much so the cell suffers from the expression (VirA is a membrane protein). This version exists in a pSB1C3 and a pSB1AT3 vector. We used the BioBrick in the pSB1AT3 plasmid for error-prone PCR and later in our two plasmid screening system together with the BBa_K389011 VirA-screening device.

virA screening device, BBa_K389011:

J23110
virG
K389002

B0017
Pvir
K389003
kanR
K389005
composite

This BioBrick contains the new, mutated virG which works in E. coli without the rpoA gene from A. tumefaciens which is expressed constitutively and a kanamycin resistance gene which is under the control of a vir promoter. It is used to screen mutants of virA which are on a different plasmid in a BBa_K389010-like part and were created by error-prone PCR. The better the VirA receptor recognizes a substance the more resistant the cell is against kanamycin. 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 replicates 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, BBa_K389012:

J23110
virG
K389002

B0017
Pvir
K389003
luc
K389004
composite

This BioBrick is similar to BBa_K389011 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, BBa_K389013:

J23110
virG
K389002

B0017
Pvir
K389003
mRFP1
E1010
composite

This BioBrick is similar to BBa_K389011 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.

virA screening approach test device, BBa_K389014:
virA
K389010

K389011
composite

This BioBrick is an assembly of BBa_K389010 and BBa_K389011. It is 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, BBa_K389015:
virA
K389010

K389012
composite

This BioBrick is an assembly of BBa_K389010 and BBa_K389012. It is used for characterizing the natural VirA/G system and the vir promoter, respectively. It is also possible to demonstrate the functionality of four new created BioBricks in one device.

vir promoter characterisation part with mRFP, BBa_K389016:
virA
K389010

K389013
composite

This BioBrick is an assembly of BBa_K389010 and BBa_K389013. It is used for characterizing the natural VirA/G system and the vir promoter, respectively.

vir promoter part, BBa_K389017:
virA
K389010

J23110
virG
K389002

B0017
Pvir
K389003
composite

This is a complete VirA/G signaling system without a reporter gene, so any gene of interest can be assembled to a vir promoter. We assembled some sensitivity tuners and a luciferase gene behind this BioBrick, so we get an enhanced luciferase signal (e.g. in part BBa_K389421).

tightly controlled lac operator, BBa_K389050:
P(Lac) IQ
I14032

B0034
lacI
C0012

B0017
LacI
R0010
composite

A lac operator is combined with a lacIq so there is always enough lacI to repress the lac operator. The system is still inducible with IPTG but it has a low basal transcription (in theory). This part should be used to test the sensitivity of the luciferase BioBrick BBa_K389004 and compare it to the sensitivity of the mRFP BioBrick BBa_E1010. But the part did not work as expected - it had a very high basal expression. The problem is probably the lacI gene BBa_C0012 from the registry which has a degradation tag. Either this tag leads to that quick degradation, that the lacI repressor cannot work as a repressor anymore or the repressor itself does not work properly.

tightly controlled lac operator + luc, BBa_K389051:

K389050

B0034
luc
K389004
composite

The part BBa_K389050 is combined with a luciferase gene as reporter. But BBa_K389050 does not work as expected so this part could not be used for its original purpose (demonstrating the sensitivity of the firefly luciferase).

tightly controlled lac operator + mRFP, BBa_K389052:

K389050

B0034
mRFP1
E1010
composite

The part BBa_K389050 is combined with a mRFP gene as reporter. But BBa_K389050 does not work as expected so this part could not be used for its original purpose (comparing the sensitivity of mRFP with the sensitivity of the firefly luciferase).

weak constitutive promoter + luc, BBa_K389302:

J23103

B0034
luc
K389004
composite

Luciferase under the control of a weak constitutive promoter.

medium strong constitutive promoter + luc, BBa_K389307:

J23115

B0034
luc
K389004
composite

Luciferase under the control of a medium strong constitutive promoter.

strong constitutive promoter + luc, BBa_K389318:

J23102

B0034
luc
K389004
composite

Luciferase under the control of a strong constitutive promoter.

sensitivity tuner no. 1 without promoter + luc readout, BBa_K389401:

I746350

B0034
mRFP1
E1010

B0015

I746360

B0034
luc
K389004
new / modified

The BBa_I746370 sensitivity tuner was modified: the arabinose promoter and the GFP readout were erased from the original part and a firefly luciferase BioBrick was added as a new readout. This part can be used behind a random promoter and gives an enhanced luciferase readout signal.

sensitivity tuner no. 2 without promoter + luc readout, BBa_K389402:

I746351

B0034
mRFP1
E1010

B0015

I746360

B0034
luc
K389004
new / modified

The BBa_I746380 sensitivity tuner was modified: the arabinose promoter and the GFP readout were erased from the original part and a firefly luciferase BioBrick was added as a new readout. This part can be used behind a random promoter and gives an enhanced luciferase readout signal.

sensitivity tuner no. 3 without promoter + luc readout, BBa_K389403:

I746352

B0034
mRFP1
E1010

B0015

I746360

B0034
luc
K389004
new / modified

The BBa_I746390 sensitivity tuner was modified: the arabinose promoter and the GFP readout were erased from the original part and a firefly luciferase BioBrick was added as a new readout. This part can be used behind a random promoter and gives an enhanced luciferase readout signal.

virA reporter device with sensitivity tuner no. 1 + luc readout, BBa_K389411:

J23110
virG
K389002

B0017
Pvir
K389003

K389401
composite

This is a modified BBa_I746370 sensitivity tuner with luciferase readout behind a vir promoter. This part also contains a virG gene under the control of a constitutive promoter. The plan is to assemble a mutated virA from a BBa_K389010-like BioBrick in front of this part to get a strong readout signal in order to reach our goal - make spiciness visible. The mutated virA is generated with error-prone PCR and screened together with the BBa_K389011 Biobrick.

virA reporter device with sensitivity tuner no. 2 + luc readout, BBa_K389412:

J23110
virG
K389002

B0017
Pvir
K389003

K389402
composite

This is a modified BBa_I746380 sensitivity tuner with luciferase readout behind a vir promoter. This part also contains a virG gene under the control of a constitutive promoter. The plan is to assemble a mutated virA from a BBa_K389010-like BioBrick in front of this part to get a strong readout signal in order to reach our goal - make spiciness visible. The mutated virA is generated with error-prone PCR and screened together with the BBa_K389011 Biobrick.

virA reporter device with sensitivity tuner no. 3 + luc readout, BBa_K389413:

J23110
virG
K389002

B0017
Pvir
K389003

K389403
composite

This is a modified BBa_I746390 sensitivity tuner with luciferase readout behind a vir promoter. This part also contains a virG gene under the control of a constitutive promoter. The plan is to assemble a mutated virA from a BBa_K389010-like BioBrick in front of this part to get a strong readout signal in order to reach our goal - make spiciness visible. The mutated virA is generated with error-prone PCR and screened together with the BBa_K389011 Biobrick.

vir promoter characterisation part with sensitivity tuner no. 1 + luc readout, BBa_K389421:
VirA/G
K389017

K389401
composite

This is a modified BBa_I746370 sensitivity tuner with luciferase readout behind a vir promoter characterisation BioBrick, so it also contains a virG and an unmutated virA gene under the control of a constitutive promoter. This BioBrick is used to prove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible.

vir promoter characterisation part with sensitivity tuner no. 2 + luc readout, BBa_K389422:
VirA/G
K389017

K389402
composite

This is a modified BBa_I746380 sensitivity tuner with luciferase readout behind a vir promoter characterisation BioBrick, so it also contains a virG and an unmutated virA gene under the control of a constitutive promoter. This BioBrick is used to prove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible.

vir promoter characterisation part with sensitivity tuner no. 3 + luc readout, BBa_K389423:
VirA/G
K389017

K389403
composite

This is a modified BBa_I746370 sensitivity tuner with luciferase readout behind a vir promoter characterisation BioBrick, so it also contains a virG and an unmutated virA gene under the control of a constitutive promoter. This BioBrick is used to prove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible.

Kanamycin-resistance cassette, BBa_P1003:
kanR
P1003
from registry

This kanamycin resistance cassette BioBrick was used as a template to create BBa_K389005.

ccdB-gene, BBa_P1010:
ccdB
P1010
from registry

The ccdB gene targets the gyrase of Escherichia coli and is lethal for all E. coli strains without the gyrase mutation gyrA462 (openwetware). The ccdB BioBrick is used for the 3A-assembly as a positive selection marker.

high-copy plasmid with Amp and Tet resistance, pSB1AT3:
pSB1AT3
pSB1AT3
from registry

This plasmid was used in 3A-assemblies and as the backbone of the mutated and unmutated BBa_K389010 part.

high-copy plasmid with Cm resistance, pSB1C3:
pSB1C3
pSB1C3
from registry

This is the plasmid used to submit the BioBricks.

lac operator, BBa_R0010:
LacI
R0010
from registry

This is a lac operator which is inducible with IPTG and repressed by lacI. It was used to build a tightly regulated lac operator (BBa_K389050) by adding a lacI repressor gene under the control of the lacIq promoter.


References