Team:Hong Kong-CUHK/BIOBRICKS CONSTRUCTION
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- | <p class="aloveofthunder" style="line-height:normal">CONSTRUCTION OF PARTS AND DEVICE</p> | + | <p class="aloveofthunder" style="line-height:normal; margin-bottom:35px">CONSTRUCTION OF PARTS AND DEVICE</p> |
<p><strong>Method of construction</strong><br /> | <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> | 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> |
Revision as of 18:12, 26 September 2012
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CONSTRUCTION OF PARTS AND DEVICE Method of construction Prolonged Overlap Extension PCR (POE-PCR) ![]() Amplification of genes Prolonged overlap extension PCR Direct Transformation Constructs List of primers Primer# primer sequence
Negative Phototactic Construct for Blue Light Detection Primers 1 and 2 were used to amplify sensory rhodopsin II coding sequence from the genome of Natronomonas Pharaonis DSM 2160. Restriction sites of HindIII and BamHI were added. [remarks]
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 4, 5 were used to amplify the coding sequence of Tsr from E. coli K-12genome. Primers 6, 7 were used to amplify the promoter J23100 and J61002 backbone from biobrick BBa_J23100. All of the parts amplified were added in PCR mix with equal molar to perform overlapping PCR and the PCR product was used for direct transformation.
Positive Phototactic Construct for Blue Light Detection ![]() 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]
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]
Phototactic Construct for Orange Light Detection ![]() Primers 11, 12 were used to amplify sensory rhodopsin I (SRI) coding sequence from the genome of Halobacterium salinarum. Restriction sites of HindIII and BamHI were added. [remarks] Restriction sites of HindIII and BamHI were added before and after the SRI gene respectively in order to:
Primers 3 and 8 were used to amplify the coding sequence of HtrI from the genome of Halobacterium salinarum. A linker (GSASNGASA) that was proven not affecting the SR system [3] was added to joint SRI and HtrI. Primers 11, 7 were used to amplify the promoter J23100 and J61002 backbone from biobrick BBa_J23100. All of the parts amplified were added in PCR mix with equal molar to perform overlapping PCR and 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).
The Gene Expression System
Red Light Sensing Construct
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