Difference between revisions of "Part:BBa K929301"
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==='''Potsdam Standard Backbone'''=== | ==='''Potsdam Standard Backbone'''=== | ||
<br><br> | <br><br> | ||
− | The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method (Blanusa ''et al.'' (2010)). For this standard we designed a new cloning vector with an RFP expression cassette as insert | + | <p style="background-color: rgb(238, 221, 130); font-weight: bold;">Introduction</p> |
− | The insert | + | |
− | After that the digested vector and the PLICed insert | + | [[image:UP12_potsdam_standard_cloning_vector_overview.png|400px|left|thumb|Potsdam Standard Cloning Vector]] |
+ | |||
+ | {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right" | ||
+ | ! colspan="2" style="background:rgb(240, 20, 70);"|[https://parts.igem.org/Part:BBa_K929301 Potsdam Standard Cloning Vector] | ||
+ | |- | ||
+ | ! colspan="2"|[[Image:UP12_Potsdam_Standard.png|300px]] | ||
+ | |- | ||
+ | |'''BioBrick Nr.''' | ||
+ | |[https://parts.igem.org/Part:BBa_K929301 BBa_K929301] | ||
+ | |- | ||
+ | |'''RFC standard''' | ||
+ | |[http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard RFC 10], [http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard Potsdam Standard] | ||
+ | |- | ||
+ | |'''Requirement''' | ||
+ | |pSB1C3<br> | ||
+ | |- | ||
+ | |'''Source''' | ||
+ | |existing part:([https://parts.igem.org/Part:BBa_J04450 BBa_J04450]) | ||
+ | |- | ||
+ | |'''Submitted by''' | ||
+ | |[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012] | ||
+ | |} | ||
+ | |||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br><br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br><br> | ||
+ | <br> | ||
+ | <br><br> | ||
+ | <br> | ||
+ | The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method (Blanusa ''et al.'' (2010)). For this standard, we designed a new cloning vector with an RFP expression cassette as insert. Therefore, we expanded the prefix with an Apa I recognition site and the suffix with a Sph I recognition site. Both enzymes causing a 3’ overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that’s the only step where we have to use restriction enzymes.<br> | ||
+ | <br> | ||
+ | <p style="background-color: rgb(238, 221, 130); font-weight: bold;">Results</p> | ||
+ | <br> | ||
+ | For culturing E.colis transformed with Potsdam Standard Cloning Vector with RFP: | ||
+ | The red fluorescence of the transformed colonies is obvious after 24 h incubation at 37 °C and over night incubation at 8 °C. To speed up the cloning, you can use LED light to see red fluorescence even after 16 h incubation at 37 °C. The transformed E.colis don't need any IPTG for RFP production. For the over night culture (fig. 1), we observed that it is better to incubate them with IPTG. After centrifugation, you can see the cerry-like cell pellets (fig. 3). | ||
+ | <br> | ||
+ | [[Image:UP12_RFP_overnightculture.png|left|400px|thumb|Fig. 1: Over night culture of Potsdam Standard Vector with RFP]] | ||
+ | [[Image:UP12_RFP_centrifugation.jpg|right|400px|thumb|Fig. 2: Red fluorescent cell pellets]] | ||
+ | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> | ||
+ | <p style="background-color: rgb(238, 221, 130); font-weight: bold;">Experimental Design: How to clone with the Potsdam Standard?</p> | ||
+ | <br> | ||
+ | The insert is amplified with primers that contain 4 phosphothioate nuclheotides and the recognition sites for Apa I and Sph I at the 5’ end. After incubation in an iodine/ethanol solution, the thiophosphates were cut out resulting in a 3’ overhang which is suitable to the overhangs that was created by cutting the Potsdam Standard vector. | ||
+ | After that, the digested vector and the PLICed insert are mixed and transformed into <i>E. coli</i> (Summary: fig. 1). By using the RFP expression cassette (fig. 2), we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.<br><br> | ||
+ | |||
+ | [[Image:UP12_Potsdam_standard.png|center|400px|thumb|Fig. 3: Summary of the experimental design]] | ||
+ | <br> | ||
+ | <!-- Add more about the biology of this part here | ||
+ | ===Usage and Biology=== | ||
+ | |||
+ | <!-- --> | ||
+ | <span class='h3bb'>Sequence and Features</span> | ||
+ | <partinfo>BBa_K929301 SequenceAndFeatures</partinfo> | ||
+ | |||
+ | |||
+ | <!-- Uncomment this to enable Functional Parameter display | ||
+ | ===Functional Parameters=== | ||
+ | <partinfo>BBa_K929301 parameters</partinfo> | ||
+ | <!-- --> |
Latest revision as of 12:54, 28 September 2012
Potsdam Standard Backbone
Introduction
Potsdam Standard Cloning Vector | |
---|---|
BioBrick Nr. | BBa_K929301 |
RFC standard | [http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard RFC 10], [http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard Potsdam Standard] |
Requirement | pSB1C3 |
Source | existing part:(BBa_J04450) |
Submitted by | [http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012] |
The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method (Blanusa et al. (2010)). For this standard, we designed a new cloning vector with an RFP expression cassette as insert. Therefore, we expanded the prefix with an Apa I recognition site and the suffix with a Sph I recognition site. Both enzymes causing a 3’ overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that’s the only step where we have to use restriction enzymes.
Results
For culturing E.colis transformed with Potsdam Standard Cloning Vector with RFP:
The red fluorescence of the transformed colonies is obvious after 24 h incubation at 37 °C and over night incubation at 8 °C. To speed up the cloning, you can use LED light to see red fluorescence even after 16 h incubation at 37 °C. The transformed E.colis don't need any IPTG for RFP production. For the over night culture (fig. 1), we observed that it is better to incubate them with IPTG. After centrifugation, you can see the cerry-like cell pellets (fig. 3).
Experimental Design: How to clone with the Potsdam Standard?
The insert is amplified with primers that contain 4 phosphothioate nuclheotides and the recognition sites for Apa I and Sph I at the 5’ end. After incubation in an iodine/ethanol solution, the thiophosphates were cut out resulting in a 3’ overhang which is suitable to the overhangs that was created by cutting the Potsdam Standard vector.
After that, the digested vector and the PLICed insert are mixed and transformed into E. coli (Summary: fig. 1). By using the RFP expression cassette (fig. 2), we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 699
Illegal AgeI site found at 811 - 1000COMPATIBLE WITH RFC[1000]