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Team:FHS Frederick MD/Sequencing
From 2014hs.igem.org
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=Sequencing= | =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. | ||
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| + | The following sequence alignment shows consensus among the engineered LOV domain and the two cloned plasmid inserts (002-027-06 and 002-027-08). | ||
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CLUSTAL 2.1 multiple sequence alignment | 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 | + | LOV GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 64 |
| - | + | 02-027-08-VF2_D11.ab1 GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 150 | |
| - | + | 02-027-06-VF2_C11.ab1 GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 149 | |
| - | + | ************************************************** | |
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| - | 02-027-08-VF2_D11.ab1 | + | |
| - | 02-027-06-VF2_C11.ab1 | + | |
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| - | LOV | + | LOV AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 114 |
| - | 02-027-08-VF2_D11.ab1 | + | 02-027-08-VF2_D11.ab1 AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 200 |
| - | 02-027-06-VF2_C11.ab1 | + | 02-027-06-VF2_C11.ab1 AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 199 |
| - | + | ************************************************** | |
| - | LOV | + | LOV ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 164 |
| - | 02-027-08-VF2_D11.ab1 | + | 02-027-08-VF2_D11.ab1 ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 250 |
| - | 02-027-06-VF2_C11.ab1 | + | 02-027-06-VF2_C11.ab1 ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 249 |
| - | + | ************************************************** | |
| - | LOV | + | LOV TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 214 |
| - | 02-027-08-VF2_D11.ab1 | + | 02-027-08-VF2_D11.ab1 TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 300 |
| - | 02-027-06-VF2_C11.ab1 | + | 02-027-06-VF2_C11.ab1 TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 299 |
| - | + | ************************************************** | |
| - | LOV | + | LOV CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 264 |
| - | 02-027-08-VF2_D11.ab1 | + | 02-027-08-VF2_D11.ab1 CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 350 |
| - | 02-027-06-VF2_C11.ab1 | + | 02-027-06-VF2_C11.ab1 CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 349 |
| - | + | ************************************************** | |
| - | LOV | + | LOV CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 314 |
| - | 02-027-08-VF2_D11.ab1 | + | 02-027-08-VF2_D11.ab1 CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 400 |
| - | 02-027-06-VF2_C11.ab1 | + | 02-027-06-VF2_C11.ab1 CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 399 |
| - | + | ************************************************** | |
| - | LOV | + | LOV CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 364 |
| - | 02-027-08-VF2_D11.ab1 | + | 02-027-08-VF2_D11.ab1 CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 450 |
| - | 02-027-06-VF2_C11.ab1 | + | 02-027-06-VF2_C11.ab1 CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 449 |
| - | + | ************************************************** | |
| - | LOV | + | LOV AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 414 |
| - | 02-027-08-VF2_D11.ab1 | + | 02-027-08-VF2_D11.ab1 AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 500 |
| - | 02-027-06-VF2_C11.ab1 | + | 02-027-06-VF2_C11.ab1 AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 499 |
| - | + | ************************************************** | |
| - | LOV | + | LOV CAAAAAGAATACGAAAAACTGCTGGAATAATACTAGTAGCGGCCGCTGCA 464 |
| - | 02-027-08-VF2_D11.ab1 | + | 02-027-08-VF2_D11.ab1 CAAAAAGAATACGAAAAACTGCTGGAATAATACTAGTAGCGGCCGCTGCA 550 |
| - | 02-027-06-VF2_C11.ab1 | + | 02-027-06-VF2_C11.ab1 CAAAAAGAATACGAAAAACTGCTGGAATAATACTAGTAGCGGCCGCTGCA 549 |
| - | + | ************************************************** | |
| - | LOV | + | LOV G---------GAAG---------------------------------AAA 472 |
| - | 02-027-08-VF2_D11.ab1 | + | 02-027-08-VF2_D11.ab1 GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAA 600 |
| - | 02-027-06-VF2_C11.ab1 | + | 02-027-06-VF2_C11.ab1 GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAA 599 |
| + | * *** *** | ||
</pre> | </pre> | ||
| + | {{:Team:FHS_Frederick_MD/Footer}} | ||
Latest revision as of 01:31, 21 June 2014
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 (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
* *** ***
