Difference between revisions of "Part:BBa K786002:Design"
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− | ===Design Notes=== | + | ===Design and construction Notes=== |
− | + | <p><strong>Method of construction</strong><br /> | |
− | + | Our team made use of a fast and convenient assembly method developed recently [1] to construct all of our biobricks in an effective way without the use of restriction enzymes and ligase - the direct transformation of prolonged overlap extension PCR products.</p> | |
− | + | https://static.igem.org/mediawiki/2012/d/d0/Con1.png | |
− | + | ||
− | + | ||
− | + | <p><strong>Amplification of genes</strong><br /> | |
− | The | + | Linear fragment DNA of the insert(s) and vector were amplified from corresponding templates by using specially designed primers which can add overlapping regions (40 bps per linear DNA) onto the DNA fragments.</p> |
+ | <p><strong>Prolonged overlap extension PCR</strong><br /> | ||
+ | Equal molar of insert(s) and vector DNA were added into a PCR reaction mix. The POE-PCR was conducted as follows: denaturation at 98°C for 30 s; 25 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 2.5 min.</p> | ||
+ | <p><strong>Direct Transformation</strong><br /> | ||
+ | Five microliter of the prolonged overlap extension PCR products was used to transform competent cells directly.</p> | ||
+ | <p><strong>Constructs</strong></p> | ||
+ | <p><strong>List of primers</strong></p> | ||
+ | <p>Primer# primer sequence</p> | ||
+ | <ol> | ||
+ | <li>TGAAAGAGGAGAAATACTAGAAGCTTATGGTGGGACTTACGACCCT</li> | ||
+ | <li>CGCCGACGCGCCGTTCGACGCGGATCCGTCGGCGACCGCAGGCGTGT</li> | ||
+ | <li>GGATCCGCGTCGAACGGCGCGTCGGCGATGTCGCTGAACGTATCACG</li> | ||
+ | <li>TGCGCCAGTCGGTGCGGACAACCGTCGGTGATGTGCGCAA</li> | ||
+ | <li>CTACACTAGCACTATCAGCGTTAAAATGTTTCCCAGTTCT</li> | ||
+ | <li>AGAACTGGGAAACATTTTAACGCTGATAGTGCTAGTGTAG</li> | ||
+ | <li>AGGGTCGTAAGTCCCACCATAAGCTTCTAGTATTTCTCCTCTTTCA</li> | ||
+ | <li>TCGCGGACATGAGTGACGGTTGTCCGCACCGACTGGCGCA</li> | ||
+ | <li>TGCGCCAGTCGGTGCGGACAACCGTCACTCATGTCCGCGA</li> | ||
+ | <li>CTACACTAGCACTATCAGCGTCAAAATGTTTCCCAGTTTG</li> | ||
+ | <li>CAAACTGGGAAACATTTTGACGCTGATAGTGCTAGTGTAG</li> | ||
+ | <li>TGAAAGAGGAGAAATACTAGAAGCTTATGGACGCCGTCGCAACCGC</li> | ||
+ | <li>TGCGCCAGTCGCTTCGTGGCACCGTCACTCATGTCCGCGA</li> | ||
+ | <li>ATTCGCGGCCGCTTCTAGAGTCCCTTGCATTTACATTTTG</li> | ||
+ | <li>ATCTAGTATTTCTCCTCTTTAGTCCATTCTCCCCAAAAAT</li> | ||
+ | <li>CTAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGT</li> | ||
+ | <li>CAAAATGTAAATGCAAGGGACTCTAGAAGCGGCCGCGAAT</li> | ||
+ | <li>GGAAAGAGGAGAAATACTAGATGGCCACCACCGTACAACT</li> | ||
+ | <li>CTAATGATGATGATGATGATGCCCTTCTTTTGTCATGCCCT</li> | ||
+ | <li>CATCATCATCATCATCATTAGTACTAGTAGCGGCCGCTGCA</li> | ||
+ | <li>ATCTAGTATTTCTCCTCTTTCCGGACCGCAGGCTGGCTAG</li> | ||
+ | </ol> | ||
+ | |||
+ | <p><strong>Positive Phototactic Construct for Blue Light Detection</strong><br /> | ||
+ | BBa_K786002<br /> | ||
+ | https://static.igem.org/mediawiki/2012/7/70/Con3.png | ||
+ | <strong> </strong><br /> | ||
+ | Primers 1 and 2 were used to amplify sensory rhodopsin II (SRII) coding sequence from the genomic DNA of <em>Natronomonas Pharaonis</em> DSM 2160. Restriction sites of HindIII and BamHI were added. [remark 1]<br /> | ||
+ | Restriction sites of HindIII and BamHI were added before and after the SRII gene respectively in order to:</p> | ||
+ | <ol> | ||
+ | <li>Enable further integration of other peptides such as His-tag, or construct a larger fusion protein (HindIII for N-terminus ligation while BamHI for C-terminus).</li> | ||
+ | <li>Enable us to switch the sensory rhodopsin portion of the fusion protein. A series of mutant sensory rhodopsins were identified which cover a large variation of absorption spectrum [2]. These two restriction sites allow further switching of the sensing unit, so the light sensing system can be tuned for sensing different kinds of light source.</li> | ||
+ | </ol> | ||
+ | <p>Primers 3, 8 were used to amplify the coding sequence of HtrII from the genome of <em>Natronomonas Pharaonis</em> DSM 2160. A linker (GSASNGASA) that was proven not affecting the SR system [3] was added to joint SRII and HtrII.</p> | ||
+ | <p>Primers 9, 10 were used to amplify the coding sequence of Tar from <em>E. coli </em>K-12 genomic DNA.</p> | ||
+ | <p>Primers 7, 11 were used to amplify the promoter J23100 and J61002 backbone from biobrick BBa_J23100.</p> | ||
+ | <p>All of the parts amplified were added into a single PCR mix with equal molar to perform overlapping PCR. The PCR product was used for direct transformation.</p> | ||
+ | <p>The insert was later on switched to pSB1C3 backbone by using EcoRI and PstI restriction enzymes and T4 ligase. [Remark 2]<br /> | ||
+ | The SpeI site after the promoter was kept so that the constitutive promoter can be switched to strictly controlled promoters such as Ptet (tetracycline-inducible promoter) and PBAD (arabinose-inducible promoter). </p> | ||
===Source=== | ===Source=== |
Revision as of 19:19, 26 September 2012
Sensory rhodopsin II (SRII) with HtrII & Tar, sensitive to blue-green light
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 37
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal SpeI site found at 37 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 785
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 37
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 37
Illegal NgoMIV site found at 140
Illegal NgoMIV site found at 398
Illegal AgeI site found at 1703 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1139
Illegal SapI site found at 913
Design and construction Notes
Method of construction
Our team made use of a fast and convenient assembly method developed recently [1] to construct all of our biobricks in an effective way without the use of restriction enzymes and ligase - the direct transformation of prolonged overlap extension PCR products.
Amplification of genes
Linear fragment DNA of the insert(s) and vector were amplified from corresponding templates by using specially designed primers which can add overlapping regions (40 bps per linear DNA) onto the DNA fragments.
Prolonged overlap extension PCR
Equal molar of insert(s) and vector DNA were added into a PCR reaction mix. The POE-PCR was conducted as follows: denaturation at 98°C for 30 s; 25 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 2.5 min.
Direct Transformation
Five microliter of the prolonged overlap extension PCR products was used to transform competent cells directly.
Constructs
List of primers
Primer# primer sequence
- TGAAAGAGGAGAAATACTAGAAGCTTATGGTGGGACTTACGACCCT
- CGCCGACGCGCCGTTCGACGCGGATCCGTCGGCGACCGCAGGCGTGT
- GGATCCGCGTCGAACGGCGCGTCGGCGATGTCGCTGAACGTATCACG
- TGCGCCAGTCGGTGCGGACAACCGTCGGTGATGTGCGCAA
- CTACACTAGCACTATCAGCGTTAAAATGTTTCCCAGTTCT
- AGAACTGGGAAACATTTTAACGCTGATAGTGCTAGTGTAG
- AGGGTCGTAAGTCCCACCATAAGCTTCTAGTATTTCTCCTCTTTCA
- TCGCGGACATGAGTGACGGTTGTCCGCACCGACTGGCGCA
- TGCGCCAGTCGGTGCGGACAACCGTCACTCATGTCCGCGA
- CTACACTAGCACTATCAGCGTCAAAATGTTTCCCAGTTTG
- CAAACTGGGAAACATTTTGACGCTGATAGTGCTAGTGTAG
- TGAAAGAGGAGAAATACTAGAAGCTTATGGACGCCGTCGCAACCGC
- TGCGCCAGTCGCTTCGTGGCACCGTCACTCATGTCCGCGA
- ATTCGCGGCCGCTTCTAGAGTCCCTTGCATTTACATTTTG
- ATCTAGTATTTCTCCTCTTTAGTCCATTCTCCCCAAAAAT
- CTAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGT
- CAAAATGTAAATGCAAGGGACTCTAGAAGCGGCCGCGAAT
- GGAAAGAGGAGAAATACTAGATGGCCACCACCGTACAACT
- CTAATGATGATGATGATGATGCCCTTCTTTTGTCATGCCCT
- CATCATCATCATCATCATTAGTACTAGTAGCGGCCGCTGCA
- ATCTAGTATTTCTCCTCTTTCCGGACCGCAGGCTGGCTAG
Positive Phototactic Construct for Blue Light Detection
BBa_K786002
Primers 1 and 2 were used to amplify sensory rhodopsin II (SRII) coding sequence from the genomic DNA of Natronomonas Pharaonis DSM 2160. Restriction sites of HindIII and BamHI were added. [remark 1]
Restriction sites of HindIII and BamHI were added before and after the SRII gene respectively in order to:
- Enable further integration of other peptides such as His-tag, or construct a larger fusion protein (HindIII for N-terminus ligation while BamHI for C-terminus).
- Enable us to switch the sensory rhodopsin portion of the fusion protein. A series of mutant sensory rhodopsins were identified which cover a large variation of absorption spectrum [2]. These two restriction sites allow further switching of the sensing unit, so the light sensing system can be tuned for sensing different kinds of light source.
Primers 3, 8 were used to amplify the coding sequence of HtrII from the genome of Natronomonas Pharaonis DSM 2160. A linker (GSASNGASA) that was proven not affecting the SR system [3] was added to joint SRII and HtrII.
Primers 9, 10 were used to amplify the coding sequence of Tar from E. coli K-12 genomic DNA.
Primers 7, 11 were used to amplify the promoter J23100 and J61002 backbone from biobrick BBa_J23100.
All of the parts amplified were added into a single PCR mix with equal molar to perform overlapping PCR. The PCR product was used for direct transformation.
The insert was later on switched to pSB1C3 backbone by using EcoRI and PstI restriction enzymes and T4 ligase. [Remark 2]
The SpeI site after the promoter was kept so that the constitutive promoter can be switched to strictly controlled promoters such as Ptet (tetracycline-inducible promoter) and PBAD (arabinose-inducible promoter).
Source
From genomic sequence of bacterial N. pharaonis (DSM 2160) and E.coli K12.