Difference between revisions of "Team:Kingsborough NY/BioBrick"

Replace this with text describing the construction of our BioBricks (3.1 and 3.2) - in other words, the colony PCR and sequencing Data

PCR to Generate Fragments for Gibson Assembly

Fragments obtained from IDT to construct our plasmid designs were not suitable for Gibson Assembly. Therefore, we ordered primers and performed PCR to generate fragments with homology to each other and/or the pSB1C3 plasmid backbone. These fragments were resolved on an agarose gel (shown on the right).

The first lane contains our molecular weight marker: Lambda DNA digested with EcoRI and PstI. the first small yellow box shows amplification of a single fragment containing all the genes for construct K2268006 (MazF and cI repressor). The second yellow box shows the various fragments (representing LacZ-alpha, cI repressor, or MazF-ssrA with appropriate homologies) that were subsequently used in Gibson Assembly (after purification, of course!).

Colony PCR to Verify Clone is Correct

For construct K2268004, the Gibson Assembly was apparently successful, and multiple clones were obtained. The Gibson control reaction and transformation control also worked. These clones were amplified using the standard iGEM sequencing primers, VF2 and VR, which have homology to the pSB1C3 backbone.

The first lane contains a successful colony PCR, giving a band that corresponds in size to the genes of construct K2268004 (compare with similar band from previous section). The second band is our molecular weight marker, Lambda DNA digested with EcoRI and PstI (prepared in-house).

Sequencing Verification

Text here about the PCR reactions to create the fragments for Gibson Assembly

Full sequencing results for construct K2268004

VF2 Primer result, reverse complement

GGGNACGTTACGTTGCGCATTTAACTATTAGATGCGTCGAATCACGAGGCAGGAATTTCAGATAAAAAAAATCCTTAGCT TTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTG ATAATGGTTGCGCTAGCAAAGAGGAGAAAGCTTATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAACTCGTGAC TGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGC CCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAAT

3.2_LinkR Primer result, reverse complement

ATCACCAGCTCCCGTCTTTCATTGCATACGAAATTCCGGAGAGCATTCATCAGGCGGGCA AGAAATGTGAATAAAGGCCGAATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAA GGCCGTAATATCCAACTGAACGGTCTGGTTATAGGTACATTGAGCACTGACTGAAATGCC CTCAAAATGTTCTTTACGATGCCATTGGGATATATCACCGGTGGTATATCCAGTGATTTT TTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGG TAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAG TGCCACCTGACGTCTAAGAACCCATTATTATCGTGACATTAACCTATAAAAATAGGCGTA TCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGA ATTCGCGGCCGCTTCTAGAGTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGAT AATGGTTGCGCTAGCAAAGAGGAGAAAGCTTATGACCATGATTACGGATTCACTGGCCGT CGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGC ACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCA ACAGAGGGCAGCTTGAATGGCGAATGGGCTTTGCCTGGTTTTTCGAATTCTTCCGCAACT TACTGCGTAGCGNA

3.2_LinkF Primer result

NTCCATGATAAGTACTCGCACTTTCTCAGCAGTCATCAGTTCGCCGCGCCAAACGTCTCTTCAGGCCACTGACTAGCGAT AACTTTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTGGGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGAC CGCTATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCAAGTCTGGCTATGCAGAAATCACCTGGCTCAACAGCC TGCTCAGGGTCAACGAGAATTAACATTCCGTCAGGAAAGCTTGGCTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTC AACCTCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGCTTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTC TAAGCTAAGGTGAGAACATCCCTGCCTGAACATGAGAAAAAACAGGGTACTCATACTCACTTCTAAGTGACGGCTGCATA CTAACCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGGGCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGC AAGCAATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAATAAAGCACCAACGCCTGACTGCCCCATCCCCATCT TGTCTGCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTTTTCATAAATTGCTTTAAGGCGACGTGCGTCCTCA AGCTGCTCTTGTGTTAATGGTTTCTTTTTTGTGCTCATAAGCTTTCTTCCTCTTTGCTAGCTGTGCTCAGTATCTTGTTA TCCGCTCACAATTTACTAGTAGCGGCCGCTGCAGGCCCGGCAAAAAAGGCAAGGGGTCACCACCCTGCCCTTTTTCTTTA AAACCGAAAAGATTACTTCGCGTTATGCAGGGTTCCTCGCTCACTGACTCGCTGCGCTCGGGCGTCGGCTGCGGCGGAGC GGATCAGCTCACTCAAGCGGTAATACGGTATCACAGAATCAGGGATACCGCAGAAGAACATGTGAGCAAAGGCAGCAAAG GCAGGACTCGTAAAAGCCGCGTGCTGGCGATTCACAAGGCTCGCCCCCTGACGGCACTACAAAATCGACGCTCTAGGCAG AGGTGCGAACCGCACGGACATAAAGGATCCAGC

* VR Primer result, reverse complement

ATGATTCGGATCCCTGGCCGTGTTTACACGTTCGGAACTGGGAAACCCTGCGTACCAACT AATCCCTTGCAGCACATCCCTTTCGCAGCTGCGTAATAGCGAAGAGGCCGCCCGATCGCC CTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGCGCTTGCCTGGTTTCCGGCACCAGAA GCGGTGCCGAAAGCTGGCTGGAGTAATATTATTACGCCACCAGCGCATAGTTTTCATCGT TCGCGCGCAAACGTCTCTTCAGGCACTGACTAGCGATAACTTTCCCCACAACGGAACACC TCTCATTGCATGGGATCATTGGGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGC TATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCAAGTCTGGCTATGCAGAAAT CACCTGGCTCAACAGCCTGCTCAGGGTCAACGAGAATTAACATTCCGTCAGGAAAGCTTG GCTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACCTCAAGCCAGAATGCAGAAT CACTGGCTTTTTTGGTTGTGCTTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAA GCTAAGGTGAGAACATCCCTGCCTGAACATGAGAAAAAACAGGGTACTCATACTCACTTC TAAGTGACGGCTGCATACTAACCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAG GGCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGCAAGCAATGCGGCGTTATAAGCAT TTAATGCATTGATGCCATTAAATAAAGCACCAACGCCTGACTGCCCCATCCCCATCTTGT CTGCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTTTTCATAAATTGCTTTAA GGCGACGTGCGTCCTCAAGCTGCTCTTGTGTTAATGGTTTCTTTTTTGTGCTCATAAGCG TTAGTATAACATTGAAGAGCTATTCGCATCACACGGATCTCCGAGCGCATTGAGCCATTG AACGAGGAATCGGAAGAGCGCCCATTAATCAATCGCCCCTCGCGCGGGGAGAGGCGGTTT GCGTATTGGGCGCTCTGCCCCTCACTCGCTCCCTCTGGAAACTGCGGCAGTACGTTGCGC GGAATGTAAGG

The sequencing primers that were used included the 3.2 Link F Primer (ggtgccggaaagctggctggagtaataaTtattacgccaccagcgcatag) and 3.2 Link R Primer (ctatgcgctggtggcgtaataAttattactccagccagctttccggcacc)