Team:LMU-Munich/Data/Vectors

From 2012.igem.org

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[[Image:BacillusBioBrickBox.png|100px|right|link=Team:LMU-Munich/Team:LMU-Munich/Bacillus_BioBricks]]
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==''Bacillus subtilis'' vectors==
==''Bacillus subtilis'' vectors==
For the detailed protocols, please see our [https://2012.igem.org/Team:LMU-Munich/Lab_Notebook/Protocols protocol section].
For the detailed protocols, please see our [https://2012.igem.org/Team:LMU-Munich/Lab_Notebook/Protocols protocol section].
-
 
===Overview===
===Overview===
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|''lacA''
|''lacA''
|empty
|empty
-
|in work
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|in progress
|not tested
|not tested
|-
|-
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|Cm
|Cm
|''amyE''
|''amyE''
-
|with <i>lacZ</i> reporter gene
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|<i>lacZ</i> reporter
|[[File:BoxChecked.png|40px|left]]
|[[File:BoxChecked.png|40px|left]]
|[[File:BoxChecked.png|40px|left]]
|[[File:BoxChecked.png|40px|left]]
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|Cm
|Cm
|''sacA''
|''sacA''
-
|with <i>luxABCDE</i> reporter cassette
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|<i>luxABCDE</i> reporter
|[[File:BoxChecked.png|40px|left]]
|[[File:BoxChecked.png|40px|left]]
|[[File:BoxChecked.png|40px|left]]
|[[File:BoxChecked.png|40px|left]]
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|Spec
|Spec
|''thrC''
|''thrC''
-
|with Xylose-inducible promoter
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|Xylose-promoter
|[[File:BoxChecked.png|40px|left]]
|[[File:BoxChecked.png|40px|left]]
|refuses transformation
|refuses transformation
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|Kan
|Kan
|replicative
|replicative
-
|with IPTG inducible promoter
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|IPTG-promoter
|[[File:BoxChecked.png|40px|left]]
|[[File:BoxChecked.png|40px|left]]
|unexpected
|unexpected
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|-
|-
|}
|}
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<div class="box">
<div class="box">
===pSB<sub>''Bs''</sub>1C===
===pSB<sub>''Bs''</sub>1C===
-
<p align="justify">
 
[[File:LMU-Munich-PSBBs1C.png|400px|left]]  
[[File:LMU-Munich-PSBBs1C.png|400px|left]]  
-
pSB<sub>Bs</sub>1C is one of our empty vectors. It integrates into the ''amy''E locus of ''Bacillus subtilis'' and carries a Chloramphenicol resistance as well as an Ampicillin resistance for ''E. coli''. Inbetween the two recombination sites, there is the constitutively expressed Cm resistance and the multiple cloning site which contains RFP to simplify the insertion of your BioBrick.
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<p align="justify">
 +
pSB<sub>Bs</sub>1C is one of our empty vectors. It integrates into the ''amy''E locus of ''Bacillus subtilis'' and carries a chloramphenicol resistance as well as an ampicillin resistance for ''E. coli''. In between the two recombination sites, there is the constitutively expressed Cm resistance and the multiple cloning site which contains RFP to simplify the insertion of your BioBrick.</p>
[[Image:LMU Firstspore.jpg|280px|link=Team:LMU-Munich/Spore_Coat_Proteins|left]][[File:LMU-Munich-starchplate.JPG | 300px|right]]
[[Image:LMU Firstspore.jpg|280px|link=Team:LMU-Munich/Spore_Coat_Proteins|left]][[File:LMU-Munich-starchplate.JPG | 300px|right]]
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The plasmid is then transformed into ''B. subtilis'' and checked via the resistance for "integration at all" and via a starch test for "integration into the right locus". Please note that the colonies without a bright surounding are correct.
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<p align="justify">
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The plasmid is then transformed into ''B. subtilis'' and checked via the resistance for "integration at all" and via a starch test for "integration into the right locus". Correct integration results in loss of halo due to disruption of the amylase gene.
-
We tested this vector with various inserts, for example the final construct of our Sporobeads with GFP. The starch test shows that the insertion in in the ''amy''E locus and fluorescence microscopy reveals the functionality of the construct.
+
</p>
 +
<p align="justify">
 +
We tested this vector with various inserts, for example the final construct of our Sporobeads with GFP. The starch test demonstrated that the plasmid inserted correctly in the ''amy''E locus and fluorescence microscopy revealed the functionality of the construct.
</p>
</p>
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<div class="box">
<div class="box">
===pSB<sub>''Bs''</sub>4S===
===pSB<sub>''Bs''</sub>4S===
-
<p align="justify">
+
 
[[File:LMU-Munich-PSBBs4S.png|400px|left]]
[[File:LMU-Munich-PSBBs4S.png|400px|left]]
[[File:LMU-Munich-Thrplate.jpg | 300px|left]]
[[File:LMU-Munich-Thrplate.jpg | 300px|left]]
-
pSB<sub>Bs</sub>4S also is an empty vector with only the multiple cloning site and the spectinomycine resistence gene in between the homologous regions. This vector integrates into the ''thr''C locus of ''B. subtilis''.  
+
<p align="justify">
-
 
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pSB<sub>Bs</sub>4S is also an empty vector with only the multiple cloning site and the spectinomycin resistance gene in between the homologous regions. This vector integrates into the ''thr''C locus of ''B. subtilis''. </p>
-
We tested the integration of this plasmid with the threonine test and a construct from the '''Suicide''' switch as insert.
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<p align="justify">
 +
We tested the integration of this plasmid with the threonine test and a construct from the '''Suicide'''switch as an insert.
<br>
<br>
<br>
<br>
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<div class="box">
<div class="box">
===pSB<sub>''Bs''</sub>2E===
===pSB<sub>''Bs''</sub>2E===
-
<p align="justify">
 
[[File:LMU-Munich-PSBBs2E.png|400px|left]]
[[File:LMU-Munich-PSBBs2E.png|400px|left]]
-
pSB<sub>Bs</sub>2E is our third empty vector, aiming to provide a set of three empty vectors with three different integration loci and three different resistances for full flexibility in their combinations.
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<p align="justify">
-
 
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pSB<sub>Bs</sub>2E is our third empty vector, aiming to provide a set of three empty vectors with three different integration loci and three different compatible resistance casseettes for full flexibility in their combination.
-
This vector is still in work. By now, only the multiple cloning site is missing. Of course, it is not tested yet.
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</p>
 +
<p align="justify">
 +
This vector is still a work in progress, but currently only the multiple cloning site is missing. Of course, it is not tested yet.
</p>
</p>
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<div class="box">
<div class="box">
===pSB<sub>''Bs''</sub>1C-<i>lac</i>Z===
===pSB<sub>''Bs''</sub>1C-<i>lac</i>Z===
-
<p align="justify">
 
[[File:LMU-Munich-PSBBs1C-lacZ.png|400px|left]]
[[File:LMU-Munich-PSBBs1C-lacZ.png|400px|left]]
[[File:Englisch_Auswertung_PliaI.png|right|300px]]
[[File:Englisch_Auswertung_PliaI.png|right|300px]]
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pSB<sub>Bs</sub>1C-<i>lac</i>Z is a reporter vector which includes a ribosome binding site, a β-galactosidase (''lac''Z) and a terminator downstream of the multiple cloning site. Also, a chloramphenicol resistance is in between the two flanking homolgy regions. This vector integrates into the ''amy''E locus which is tested by the starch test.
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<p align="justify"> pSB<sub>Bs</sub>1C-<i>lac</i>Z is a reporter vector, which contains the multiple cloning site upstream of a functional reporter device, consisting of ribosome binding site, β-galactosidase gene ''lac''Z and terminator. Also, a chloramphenicol resistance is in between the two flanking homolgy regions. Upon chloramphenicol selection, this vector integrates into the ''amy''E locus, which can be verified by the starch test.</p>
-
 
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<p align="justify">
-
This vector was used for several promoter evaluations.
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This vector was used for several [[Team:LMU-Munich/Data#Anderson_Promoter_Evaluation |promoter evaluations]].
</p>
</p>
</div>
</div>
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[[File:LMU-Munich-PSBBs3C-luxABCDE.png|400px|left]]
[[File:LMU-Munich-PSBBs3C-luxABCDE.png|400px|left]]
-
<p align="justify"> pSB<sub>Bs</sub>3C-<i>luxABCDE</i> is our second reporter vector. Downstream of the multiple cloning site, there are a ribosome binding site, the ''lux''ABCDE operon and a terminator. Also, a chloramphenicol resistance is in between the two flanking homolgy regions. This vector integrates into the ''sac''A locus of ''B. subtilis'' which is tested for by colony PCR. [[Image:LMU-Munich-Gelfoto_colony_PCR.png | 300px|right]]One primer is located outside of the region and its respective partner is located on the inegrative part of the vector, facing outwards. gDNA from W168 as a negative control should not give a PCR product.
+
<p align="justify"> pSB<sub>Bs</sub>3C-<i>luxABCDE</i> is our second reporter vector. It contains the multiple cloning site upstream of a functional reporter device, consisting of the ''lux''-operon and a downstream terminator. Upon chloramphenicol selection, this vector integrates, this vector integrates into the ''sac''A locus of ''B. subtilis'' by double homologous recombination. [[Image:LMU-Munich-Gelfoto_colony_PCR.png | 300px|right]] Correct integration is verified by colony PCR. One primer is located outside the integration site and its respective partner is located on the inegrated part of the vector, facing outwards. gDNA from W168 as a negative control should not give a PCR product.</p>
<br>
<br>
-
A pre version of this vector with one in BioBrick standard forbidden PstI site was used for several promoter evaluations. All graphs that you can find in the Data section of the promoters derive from this pre version of the vector. To show that our last version of this vector also works we did another experiment where we compared the luminescence of P<sub>''lepA''</sub> in the "old" vector with the version whithout any forbidden sites. Here you can see the results of this experiment:</p>
+
<p align="justify">
-
[[File:PlepA Vektorvergleich.png|thumb|400px|center|<p align="justify">Luminescence measurements of the promoter P<sub>''lepA''</sub> in the pre version of the vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> (one PstI site left) in comparison to the last version of the vectorwithout any forbidden resriction site.</p>]]
+
A pre-version of this vector with one forbidden ''Pst''I site was used for most promoter evaluations (All graphs that you can find in the Data section of the promoters derive from this pre-version). To prove that our final version also works, we did another experiment where we compared the luminescence of P<sub>''lepA''</sub> in the pre- and final version of pSB<sub>Bs</sub>3C-<i>luxABCDE</i>. Here you can see the results of this experiment:</p>
-
In this experiment with the same promoter P<sub>''lepA''</sub> the finished vector showed about half of the activity of the pre version where there was still one forbidden PstI site left.</p>
+
[[File:PlepA Vektorvergleich.png|thumb|400px|center|<p align="justify">Luminescence measurements of the promoter P<sub>''lepA''</sub> in the pre-version of the vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> in comparison to the final vector.</p>]]
 +
<p align="justify">
 +
In this experiment, the finished vector showed about half of the activity throughout the measurement, hence while the absolute luminescence values are lower, the overall dynamics are unaffected.</p>
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<div class="box">
<div class="box">
===pSB<sub>''Bs''</sub>4S-P<sub><i>Xyl</i></sub>===
===pSB<sub>''Bs''</sub>4S-P<sub><i>Xyl</i></sub>===
-
<p align="justify"> CAUTION: DOES NOT WORK! CANNOT BE TRANSFORMED INTO ''B. SUBTILIS''
+
<p align="justify"> <font size="4" color="FF0000">CAUTION: DOES NOT WORK! CANNOT BE TRANSFORMED INTO ''B. SUBTILIS''!</font>
-
[[File:LMU-Munich-PSBBs4S-Pxyl.png|400px|left]]
+
[[File:LMU-Munich-PSBBs4S-Pxyl.png|400px|left]]</p>
-
pSB<sub>''Bs''</sub>4S-P<sub><i>Xyl</i></sub> is an integrative expression vector. Upstream of the multiple cloning site, there is P<sub>''Xyl''</sub>, a xylose-inducible promoter, and downstream there is a terminator. In absence of Xylose,  P<sub>''Xyl''</sub> is blocked by the repressor XylR which is expressed in ''B. subtilis'' and not part of this vector. Apart from that, also a spectinomycine resistance is located in between the two recombination sites. This vector integrates into the ''thrC'' locus which is checked for by the threonine test.  
+
<p align="justify">
 +
pSB<sub>''Bs''</sub>4S-P<sub><i>Xyl</i></sub> is an integrative expression vector. Upstream of the multiple cloning site, there is a xylose-inducible promoter, P<sub>''xyl''</sub>, followed by a terminator downstream. In the absence of xylose,  P<sub>''xyl''</sub> is inhibited by the repressor XylR, which is expressed in ''B. subtilis'' and not part of this vector. For selection, a spectinomycin resistance cassette is located in between the recombination sites. This vector integrates into the ''thrC'' locus, which can be checked by the threonine test. </p>
-
However, we could not manage to transform this vector into ''B. subtilis'', although we tried three times. Also the team from [https://2012.igem.org/Team:Groningen Groningen] tested this vector, but was not able to transform it into ''B. subtilis'', either.
+
<p align="justify">
 +
However, we could not manage to transform this vector into ''B. subtilis'', although we tried three times. Moreover, the team from [https://2012.igem.org/Team:Groningen Groningen] also tested this vector, but again was not able to transform it into ''B. subtilis'', either.
</p>
</p>
 +
<p align="justify">
 +
There are two possible reasons for the failure: First, a defective resistance cassette or second, the presence of a forbidden restriction site preventing correct linearization prior to transformation.</p>
</div>
</div>
<div class="box">
<div class="box">
===pSB<sub>''Bs''</sub>0K-P<sub><i>spac</i></sub>===
===pSB<sub>''Bs''</sub>0K-P<sub><i>spac</i></sub>===
-
CAUTION: THIS VECTOR DOES NOT WORK AS EXPECTED! THE PROMOTER IS STRONG CONSTITUTIVE INSTEAD OF IPTG-INDUCIBLE!
+
<font size="4" color="FF0000">CAUTION: THIS VECTOR DOES NOT WORK AS EXPECTED! THE PROMOTER IS STRONG CONSTITUTIVE INSTEAD OF IPTG-INDUCIBLE!</font>
[[File:LMU-Munich-PSBBs0K-Pspac.png|400px|left]]
[[File:LMU-Munich-PSBBs0K-Pspac.png|400px|left]]
<p align="justify">
<p align="justify">
-
pSB<sub>''Bs''</sub>0K-P<sub><i>spac</i></sub> is a replicative expression vector with a kanamycin resistance. The IPTG (isopropylbeta-D-thiogalactopyranoside)-inducible Promoter P<sub>''spac''</sub> is followed by the multiple cloning site and a terminator. Also expressed are ''lac''Y, a transporter for IPTG (naturally allolactose), and ''lac'', the repressor. In presence of IPTG, LacI releases from the promoter and the gene of interest is expressed.  
+
pSB<sub>''Bs''</sub>0K-P<sub><i>spac</i></sub> is a replicative expression vector with a kanamycin resistance. The IPTG -inducible promoter P<sub>''spac''</sub> is followed by the multiple cloning site and a terminator. Moreover, it contains ''lac''Y, encoding aβ-Galactosid-Permease, and ''lac''I, encoding the Lac-repressor. In the presence of IPTG, LacI is released from the promoter and the gene of interest is expressed.  
-
 
+
</p>
-
The ''ble'' gene encodes the bleomycin resisitance protein (BRP) which can be selected for by bleomycine or phleomycin in ''E. coli'' and ''B. subtilis''. We did not use this resistance.
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<p align="justify">
-
 
+
The ''ble'' gene encodes the bleomycin resisitance protein (BRP), which can be used for by bleomycin selection in ''E. coli'' and ''B. subtilis''. We did not use this resistance.
-
The presence of the vector can be checked for via colony PCR with the primers: CTACATCCAGAACAACCTCTGC and TTCGGAAGGAAATGATGACCTC. We did not perfom the colony PCR because we selected for functionality on plates with X-Gal and IPTG and we got blue colonies.  
+
</p>
-
 
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<p align="justify">
 +
The presence of the vector can be checked by colony PCR with the primers: CTACATCCAGAACAACCTCTGC and TTCGGAAGGAAATGATGACCTC. We did not perfom the colony PCR because we selected for functionality of our lacZ-construct (see figure below) on plates with X-Gal and IPTG, and got blue colonies.  
 +
</p>
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
| style="width: 70%;background-color: #EBFCE4;" |
| style="width: 70%;background-color: #EBFCE4;" |
{|
{|
-
|[[File:LMU-Munich-0K-OD.png|500px|center|]]
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|[[File:LMU-Munich-0K-OD.png|500px|center]]
|-
|-
| style="width: 70%;background-color: #EBFCE4;" |
| style="width: 70%;background-color: #EBFCE4;" |
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|}
|}
|}
|}
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<p align="justify">
-
This expression vector was tested by insertion of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823016 ''lac''Z] into the multiple cloning site, transformation into ''W168'' and ONPG assays. Overnight cultures were diluted 1:100 in LB-medium and incubated at 37°C, 230 rpm. Cells were harvested in 2 ml reaction tubes and frozen. For the induction assay, at an OD<sub>600</sub> around 0.9 were split into 3 ml aliquots in test tubes with the given IPTG-concentrations and incubated for another hour. Data for splitting at OD<sub>600</sub> around 0.3 is not shown, but similar.
+
This expression vector was tested by insertion of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823016 ''lac''Z] into the multiple cloning site, transformation into ''W168'' and ONPG assays. Overnight cultures were diluted 1:100 in LB-medium and incubated at 37°C, 230 rpm. Cells were harvested in 2 ml reaction tubes and frozen. For the induction assay, a culture of about OD<sub>600</sub>=0.9 was split into 3 ml aliquots in test tubes with different IPTG-concentrations and incubated for another hour. (Same results were obtained for OD<sub>600</sub>=0.3, data not shown.)
-
 
+
</p>
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
| style="width: 70%;background-color: #EBFCE4;" |
| style="width: 70%;background-color: #EBFCE4;" |
{|
{|
-
|[[File:LMU-Munich-0K-IPTG.png|500px|center|]]
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|[[File:LMU-Munich-0K-IPTG.png|500px|center]]
|-
|-
| style="width: 70%;background-color: #EBFCE4;" |
| style="width: 70%;background-color: #EBFCE4;" |
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-
 
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<p align="justify">
-
In summary, the figures show that the expression is very strong, even without induction [in comparison to the native ''B. subtilis'' promoters] and does not change with IPTG addition. It does change depending on the growth phase, with a maximum strength in the stationary phase.
+
In summary, the figures show that the expression is uniformly very strong [in comparison to the native ''B. subtilis'' promoters], even without addition of IPTG. It does change depending on the growth phase, with a maximum strength in the stationary phase.
-
 
+
</p>
-
This vector was designed for overexpression of proteins, but is not inducible but constitutively active.
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<p align="justify">
-
<p>
+
This vector was designed for the inducible overexpression of proteins, but is constitutively active.
 +
</p>
 +
<p align="justify">
 +
Possible reason for these results are: a dysfunctional Lac-repressor, or point mutations in the operator. This needs to be checked by sequencing.</p>
</div>
</div>
<div class="box">
<div class="box">
==='''Sporo'''vector===
==='''Sporo'''vector===
-
<p align="justify">
 
-
Our '''Sporo'''vector is specially designed to give you the opportunity to easily create Sporobeads with any proteins you can think of. It is available as BioBrick in pSB1C3 and can be cloned in any of our empty ''B. subtilis'' vectors. We also offer it “precloned” in pSB<sub>Bs</sub>4S which in this version lacks ''NgoM''IV restriction sites.
 
-
[[File:Sporovectorcloning.png|600px]]
 
-
The '''Sporo'''vector was created by ligation of three cut PCR-products. It is designed to allow N-terminal fusion of genes in Freiburg standard ([http://dspace.mit.edu/bitstream/handle/1721.1/45140/BBF_RFC%2025.pdf?sequence=1 RCF25]). Therefore, the gene to be inserted must be cut with ''Xba''I and ''Age''I and the vector must be digested with ''Xba''I and ''NgoM''IV. By this digest, the RFP cassette will be removed from this vector to allow easy selection for the integration of the new insert. After ligation, there is a scar of six nucleotides between both genes but no restriction site left.
 
-
The gene fusion is controlled by the P<sub>''cot''YZ</sub>-promoter which was the strongest one in our promoter evaluations. The C-terminal gene part is ''cge''A, one of the two known spore crust proteins.
 
-
The vector is cloned but has not been tested with an insert, yet.
+
<p align="justify">
-
We are working on further '''Sporo'''vectors to offer also fusion with ''cot''Z as well as C-terminal fusions.
+
Our '''Sporo'''vector is specially designed to give you the opportunity to easily create '''Sporo'''beads with any fusion protein you can think of. The part between the ''EcoR''I and ''Pst''I is available as BioBrick in pSB1C3 and can be cloned in any of our empty ''B. subtilis'' vectors. We also offer it “precloned” in pSB<sub>Bs</sub>4S, which in this version lacks the ''NgoM''IV restriction sites.
</p>
</p>
 +
[[File:Sporovectorcloning.png|600px|center]]
 +
<p align="justify">
 +
The '''Sporo'''vector was created by ligation of three cut PCR-products with special overhangs (P<sub>''cotYZ''</sub>, ''mRFP'' and ''cgeA''-B0014). It is designed to allow N-terminal fusions of genes in Freiburg standard ([http://dspace.mit.edu/bitstream/handle/1721.1/45140/BBF_RFC%2025.pdf?sequence=1 RFC25]). Towards that end, the gene to be inserted must be cut with ''Xba''I and ''Age''I and the vector must be digested with ''Xba''I and ''NgoM''IV. This removes the RFP cassette and allows easy selection for the integration of the new insert. After ligation, there is a scar of six nucleotides between both genes but no restriction site left. </p>
 +
<p align="justify">
 +
Expression of the gene fusion is controlled by the P<sub>''cot''YZ</sub>-promoter, which was the strongest in our promoter evaluations. The C-terminal part of the resulting fusion gene encodes CgeA, one of the two known spore crust proteins.
 +
</p>
 +
<p align="justify">
 +
The vector is finished but has not yet been tested with an insert.
 +
We are working on three additional '''Sporo'''vectors to also offer C and N-terminal fusions with both ''cot''Z and ''cge''A.
 +
</p>
 +
</div>
</div>

Latest revision as of 13:16, 26 October 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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