Sequencing

In the last week of school, as classes were winding down, we sent our plasmid samples out for sequencing using the BioBrick standard forward sequencing primer (FV2). Our first shot at sequencing the samples yielded poor results because our template concentration was too high.

We quickly resubmitted another set of samples and this time asked the company to adjust the template concentrations prior to sequencing. This yielded good results with mixed outcomes.

The bad news: None of the five pSB1C3/NirB transformants showed signs of having integrated the NirB promoter. We are in the process of considering what went wrong and how to get better results next time.

The good news: We were thrilled to discover that both of the pSB1C3/LOV plasmid samples had successfully incorporated our re-engineered copy of the LOV gene. While other teams have made BioBricks containing the NirB oxygen sensitive promoters ([http://parts.igem.org/Part:BBa_K763002 BBa_K763002]), we believe we were the first to create a BioBrick containing an optimized version of the LOV domain, which should have better fluorescent properties than green fluorescent protein in anaerobic environments.

The following sequence alignment shows consensus among the engineered LOV domain and the two cloned plasmid inserts (002-027-06 and 002-027-08).

CLUSTAL 2.1 multiple sequence alignment

LOV                    --------------------------------------------GAATTC 14
02-027-08-VF2_D11.ab1  TTCNGATAAAAAAAATCCTTAGCTTTCGCNANNGNNGATTTCTGGAATTC 100
02-027-06-VF2_C11.ab1  TTCAGATAAAAAAAATCCTTAGCTTTCGCNNAGGANGATTTCTGGAATTC 99
                                                                   ******

LOV                    GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 64
02-027-08-VF2_D11.ab1  GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 150
02-027-06-VF2_C11.ab1  GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 149
                       **************************************************

LOV                    AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 114
02-027-08-VF2_D11.ab1  AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 200
02-027-06-VF2_C11.ab1  AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 199
                       **************************************************

LOV                    ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 164
02-027-08-VF2_D11.ab1  ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 250
02-027-06-VF2_C11.ab1  ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 249
                       **************************************************

LOV                    TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 214
02-027-08-VF2_D11.ab1  TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 300
02-027-06-VF2_C11.ab1  TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 299
                       **************************************************

LOV                    CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 264
02-027-08-VF2_D11.ab1  CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 350
02-027-06-VF2_C11.ab1  CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 349
                       **************************************************

LOV                    CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 314
02-027-08-VF2_D11.ab1  CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 400
02-027-06-VF2_C11.ab1  CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 399
                       **************************************************

LOV                    CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 364
02-027-08-VF2_D11.ab1  CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 450
02-027-06-VF2_C11.ab1  CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 449
                       **************************************************

LOV                    AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 414
02-027-08-VF2_D11.ab1  AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 500
02-027-06-VF2_C11.ab1  AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 499
                       **************************************************

LOV                    CAAAAAGAATACGAAAAACTGCTGGAATAATACTAGTAGCGGCCGCTGCA 464
02-027-08-VF2_D11.ab1  CAAAAAGAATACGAAAAACTGCTGGAATAATACTAGTAGCGGCCGCTGCA 550
02-027-06-VF2_C11.ab1  CAAAAAGAATACGAAAAACTGCTGGAATAATACTAGTAGCGGCCGCTGCA 549
                       **************************************************

LOV                    G---------GAAG---------------------------------AAA 472
02-027-08-VF2_D11.ab1  GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAA 600
02-027-06-VF2_C11.ab1  GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAA 599
                       *          ***                                 ***