Team:FHS Frederick MD/Sequencing

From 2014hs.igem.org

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(Added LOV sequence data.)
(Added footer)
 
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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.
 +
 +
The following sequence alignment shows consensus among the engineered LOV domain and the two cloned plasmid inserts (002-027-06 and 002-027-08).
Line 11: Line 17:
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                       ------------GTTTCTTC------------------------------ 8
+
LOV                   GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 64
-
02-027-08-VF2_D11.ab1      NNNNNNNNNNNNNNNNNNNNANTANAAATAGNNNTATCACGAGGCAGAAT 50
+
02-027-08-VF2_D11.ab1 GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 150
-
02-027-06-VF2_C11.ab1      -NNNNNNNNNNNNNNNNNNNNNNAAAAATAGGCGTATCACGAGGCAGAAT 49
+
02-027-06-VF2_C11.ab1 GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 149
-
                                                                           
+
                       **************************************************
-
 
+
-
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
+
-
                          *          ***                                ***
+
-
 
+
-
LOV                        C------------------------------------------------- 473
+
-
02-027-08-VF2_D11.ab1      CCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCT 650
+
-
02-027-06-VF2_C11.ab1      CCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCT 649
+
-
                          *                                               
+
-
 
+
-
LOV                        --------------------------------------------------
+
-
02-027-08-VF2_D11.ab1      GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA-GGCG 699
+
-
02-027-06-VF2_C11.ab1      GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCNAAAGGCG 699
+
-
                                                                           
+
-
 
+
-
LOV                        --------------------------------------------------
+
-
02-027-08-VF2_D11.ab1      GTAATACGGTTATCCACAGAATCAGGGGATAACGCANGAAAGAACATGTG 749
+
-
02-027-06-VF2_C11.ab1      GTAATACGGTTATCCACAGAATCAGGGGATAACGCANGAAAGAACATGTG 749
+
-
                                                                           
+
-
LOV                       --------------------------------------------------
+
LOV                   AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 114
-
02-027-08-VF2_D11.ab1     AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGNCGCGTTGCTGG 799
+
02-027-08-VF2_D11.ab1 AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 200
-
02-027-06-VF2_C11.ab1     AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG 799
+
02-027-06-VF2_C11.ab1 AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 199
-
                                                                           
+
                      **************************************************
-
LOV                       --------------------------------------------------
+
LOV                   ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 164
-
02-027-08-VF2_D11.ab1     CGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC 849
+
02-027-08-VF2_D11.ab1 ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 250
-
02-027-06-VF2_C11.ab1     CGTTTTTCCACANGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC 849
+
02-027-06-VF2_C11.ab1 ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 249
-
                                                                           
+
                      **************************************************
-
LOV                       --------------------------------------------------
+
LOV                   TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 214
-
02-027-08-VF2_D11.ab1     TCAAGTCNNAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTT 899
+
02-027-08-VF2_D11.ab1 TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 300
-
02-027-06-VF2_C11.ab1     TCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCANGCGTT 899
+
02-027-06-VF2_C11.ab1 TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 299
-
                                                                           
+
                      **************************************************
-
LOV                       --------------------------------------------------
+
LOV                   CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 264
-
02-027-08-VF2_D11.ab1     TCCCCCTGGAAAGCTCCCTCGTGCGCTCTCCTGTTCCNACCCTGCCGCTT 949
+
02-027-08-VF2_D11.ab1 CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 350
-
02-027-06-VF2_C11.ab1     TCCCCCTGGAAAGCTCCCTCGTGCGCTCTCCTGTTCCNACCCTGCCGCTT 949
+
02-027-06-VF2_C11.ab1 CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 349
-
                                                                           
+
                      **************************************************
-
LOV                       --------------------------------------------------
+
LOV                   CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 314
-
02-027-08-VF2_D11.ab1     ACCGGATACCTGTCCGCCTTTCTCCCTTCGGGNAANCGTGGCGCTTTCTC 999
+
02-027-08-VF2_D11.ab1 CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 400
-
02-027-06-VF2_C11.ab1     ACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAA-GCGTGGCNCTTTCTC 998
+
02-027-06-VF2_C11.ab1 CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 399
-
                                                                           
+
                      **************************************************
-
LOV                       --------------------------------------------------
+
LOV                   CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 364
-
02-027-08-VF2_D11.ab1     ATAGCTCACGNNTGTAGGNATCTCAGTTCGGNGNAGGTCGTTCGCTNCNA 1049
+
02-027-08-VF2_D11.ab1 CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 450
-
02-027-06-VF2_C11.ab1     ATAGCTCACGCT-GTNNGTATCTCANTTCNGNGNNAGNCGTTCGCNNCNA 1047
+
02-027-06-VF2_C11.ab1 CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 449
-
                                                                           
+
                      **************************************************
-
LOV                       --------------------------------------------------
+
LOV                   AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 414
-
02-027-08-VF2_D11.ab1     GCTGGG-CTNNNGNGCNCGAACCCCCGTTCANNCCGACGNCTGCNCCTTA 1098
+
02-027-08-VF2_D11.ab1 AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 500
-
02-027-06-VF2_C11.ab1     NCTNGGGCTNNNGNNCANNAANNNNC------------------------ 1073
+
02-027-06-VF2_C11.ab1 AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 499
-
                                                                           
+
                      **************************************************
-
LOV                       --------------------------------------------------
+
LOV                   CAAAAAGAATACGAAAAACTGCTGGAATAATACTAGTAGCGGCCGCTGCA 464
-
02-027-08-VF2_D11.ab1     TCCNGNNANNTATNGTCTTGANTCCAACCCGGGTAANAANNNNAANTTNA 1148
+
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     NNNNCNNN 1156
+
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

FHS Logo.png


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
                       *          ***                                 ***
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