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Team:Cambridge/Project/Results
From 2012.igem.org
Contents |
Results
Set out below are the developments the team has made over the summer, in tackling our aim and objectives.
RiboSense
Ratiometrica
After a lot of technical difficulties, we were able to assemble our fluorescent construct using Gibson assembly. The photo below shows the predicted and obtained digest pattern using HINDIII.
The construct is also sequenced from the beginning and middle (as it is a long part) by Source Bioscience, below is the alignment data from the sequencing (using ClustalW2):
CLUSTAL 2.1 multiple sequence alignment
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AACAAAATTCTCCAGTCTTCACATCGGTTTGAAAGGAGGAAGCGGAAGAA 50
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TGAAGTAAGAGGGATTTTTGACTCCGAAGTAAGTCTTCAAAAAATCAAAT 100
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AAGGAGTGTCAAGAATGTTTGCAAAACGATTCAAAACCTCTTTACTGCCG 150
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTATTCGCTGGATTTTTATTGCTGTTTCATTTGGTTCTGGCAGGACCGGC 200
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GGCTGCGAGTGCTGAAACGGCGAACAAATCGAATGAGCTTACAGCACCGT 250
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CGATCAAAAGCGGAACCATTCTTCATGCATGGAATTGGTCGTTCAATACG 300
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTAAAACACAATATGAAGGATATTCATGATGCAGGATATACAGCCATTCA 350
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GACATCTCCGATTAACCAAGTAAAGGAAGGGAATCAAGGAGATAAAAGCA 400
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TGTCGAACTGGTACTGGCTGTATCAGCCGACATCGTATCAAATTGGCAAC 450
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CGTTACTTAGGTACTGAACAAGAATTTAAAGAAATGTGTGCAGCCGCTGA 500
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AGAATATGGCATAAAGGTCATTGTTGACGCGGTCATCAATCATACCACCA 550
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GTGATTATGCCGCGATTTCCAATGAGGTTAAGAGTATTCCAAACTGGACA 600
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CATGGAAACACACAAATTAAAAACTGGTCTGATCGGATCCTAGAAGCTTA 650
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCGAATTCTCATGTTTGACAGCTTATCATCGGCAATAGTTACCCTCTCTC 700
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCT 750
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT 800
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCGCCGCTCTAGCTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCG 850
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTTCTACAAACTCTTGTTAACTCTAGAGCTGCCTGCCGCGTTTCGGTGAT 900
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GAAGATCTTCCCGATGATTAATTAATTCAGAACGCTCGGTTGCCGCCGGG 950
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CGTTTTTTATGCAGCAATGGCAAGAACGTTGCTCTAGAATAATTCTACAC 1000
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AGCCCAGTCCAGACTATTCGGCACTGAAATTATGGGTGAAGTGGTCAAGA 1050
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCTCACTAGGCACCTTAAAAATAGCGCACCCTGAAGAAGATTTATTTGAG 1100
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GTAGCCCTTGCCTACCTAGCTTCCAAGAAAGATATCCTAACAGCACAAGA 1150
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCGGAAAGATGTTTTGTTCTACATCCAGAACAACCTAATTGTGAGCGCTC 1200
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ACAATTTTTTGCAAAAAGTTGTTGACTTTATCTACAAGGTGTGGCATAAT 1250
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GTGTGGAATTGTGAGCGGCTCACAATTAAAGGAGGAATTCAAAATGGTGA 1300
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTG 1350
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GACGGCGACGTGAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGG 1400
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCA 1450
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGGGCGTG 1500
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAA 1550
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGG 1600
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACC 1650
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA 1700
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACAACGTCTATA 1750
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGC 1800
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAA 1850
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGA 1900
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG 1950
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA 2000
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCTGTACAAGTAATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAA 2050
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACT 2100
Rat3 -----------------------------NNNNNNNNNNNNNNNNNNTTN 21
pJS150_seq_-_Ratiometrica AGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAAATTT 2150
... .. .... . **.
Rat3 TGNCNNN-TAATTTTATTGACAACGTCTTATTAACGTTGATATAATTTAA 70
pJS150_seq_-_Ratiometrica TGTCAAAATAATTTTATTGACAACGTCTTATTAACGTTGATATAATTTAA 2200
**.* ******************************************
Rat3 ATTTTATTTGN-NAAAATGGGCTCGTGTTGTACAATAAATGTTACTAGAG 119
pJS150_seq_-_Ratiometrica ATTTTATTTGACAAAAATGGGCTCGTGTTGTACAATAAATGTTACTAGAG 2250
********** *************************************
Rat3 AAAGGTGGTGNATACTAGATGGTGAGCAAGGGCGAGGAGCTGTTCACCGG 169
pJS150_seq_-_Ratiometrica AAAGGTGGTGAATACTAGATGGTGAGCAAGGGCGAGGAGCTGTTCACCGG 2300
********** ***************************************
Rat3 GGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGT 219
pJS150_seq_-_Ratiometrica GGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGT 2350
**************************************************
Rat3 TCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC 269
pJS150_seq_-_Ratiometrica TCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC 2400
**************************************************
Rat3 CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT 319
pJS150_seq_-_Ratiometrica CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT 2450
**************************************************
Rat3 CGTGACCACCTTCGGCTACGGCCTGCAATGCTTCGCCCGCTACCCCGACC 369
pJS150_seq_-_Ratiometrica CGTGACCACCTTCGGCTACGGCCTGCAATGCTTCGCCCGCTACCCCGACC 2500
**************************************************
Rat3 ACATGAAGCTGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC 419
pJS150_seq_-_Ratiometrica ACATGAAGCTGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC 2550
**************************************************
Rat3 CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGC 469
pJS150_seq_-_Ratiometrica CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGC 2600
**************************************************
Rat3 CGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG 519
pJS150_seq_-_Ratiometrica CGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG 2650
**************************************************
Rat3 GCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTAC 569
pJS150_seq_-_Ratiometrica GCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTAC 2700
**************************************************
Rat3 AACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGG 619
pJS150_seq_-_Ratiometrica AACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGG 2750
**************************************************
Rat3 CATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGC 669
pJS150_seq_-_Ratiometrica CATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGC 2800
**************************************************
Rat3 AGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTG 719
pJS150_seq_-_Ratiometrica AGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTG 2850
**************************************************
Rat3 CTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGA 769
pJS150_seq_-_Ratiometrica CTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGA 2900
**************************************************
Rat3 CCCCNAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCC 819
pJS150_seq_-_Ratiometrica CCCC-AACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCC 2949
**** *********************************************
Rat3 GCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATAATGATACTA 869
pJS150_seq_-_Ratiometrica GCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATAATGATACTA 2999
**************************************************
Rat3 GAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTT 919
pJS150_seq_-_Ratiometrica GAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTT 3049
**************************************************
Rat3 CGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGTCATCATTTCC 969
pJS150_seq_-_Ratiometrica CGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGTCATCATTTCC 3099
**************************************************
Rat3 TTCCGAAAAAACGGTTGCATTTAAATCTTACATATGTAATACTTTTCAAA 1019
pJS150_seq_-_Ratiometrica TTCCGAAAAAACGGTTGCATTTAAATCTTACATATGTAATACTTT-CAAA 3148
********************************************* ****
Rat3 GACTACATTTTGTAAGATTTGATGTTTGAANNCGGGCTGAAANATCGGTA 1069
pJS150_seq_-_Ratiometrica GACTACATTT-GTAAGATTTGATGTTTGAG-TCGG-CTGAAAGATCG-TA 3194
********** ******************. .*** ******.**** **
Rat3 CGTACCCNNNNTTGTTTCNNNATNNNNCAG-CCNATNNNCTNNNNGNATA 1118
pJS150_seq_-_Ratiometrica CGTACCAATTATTGTTTCGTGATTGTTCAAGCCATAACACTGTAGGGATA 3244
******. .. *******...**....**. ** :: **.. .*.***
Rat3 NNNNNNNAAGAGNNCTTCNNCNGGNNACNANTCANNNAANNANTNAANCN 1168
pJS150_seq_-_Ratiometrica GTGG--AAAGAGTGCTTCATCTGGTTACGA-TCAATCAAATATTCAAACG 3291
.... *****..**** .*.**..**.* *** . ** .*.* ** *.
Rat3 GNNGGNNNACNNNNTNNNN---ANNNNNNANNTTNNNCGAANNN------ 1209
pJS150_seq_-_Ratiometrica GAGGGAG-ACGATTTTGATGAAACCAGTAACGTTATACGATGTCGCAGAG 3340
* .** . **. ..*.. . * .. * .** . ***:..
Rat3 ----CCNNNNNNNNTNNNNNNNNGNNNNNNNNNANNN------------- 1242
pJS150_seq_-_Ratiometrica TATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAG 3390
**..... . *. . . .... . ....
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGC 3440
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCG 3490
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCA 3540
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGG 3590
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTG 3640
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCT 3690
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGG 3740
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTC 3790
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGG 3840
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGC 3890
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAG 3940
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACA 3990
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTG 4040
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGG 4090
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGA 4140
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CAGCTCATGTTATATCCCGCCGTCAACCACCATCAAACAGGATTTTCGCC 4190
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAG 4240
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAAC 4290
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATT 4340
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTAA 4390
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AAAAAGCAATGTATGGGTCTCCCCGCTACATTGCTTTTTTTATAGCTGTT 4440
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AGTTCAACAAACGAAAATTGGATAAAGTGGGATATTTTTAAAATATATAT 4490
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTATGTTACAGTAATATTGACTTTTAAAAAAGGATTGATTCTAATGAAGA 4540
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AAGCAGACAAGTAAGCCTCCTAAATTCACTTTAGATAAAAATTTAGGAGG 4590
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CATATCAAATGAACTTTAATAAAATTGATTTAGACAATTGGAAGAGAAAA 4640
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GAGATATTTAATCATTATTTGAACCAACAAACGACTTTTAGTATAACCAC 4690
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AGAAATTGATATTAGTGTTTTATACCGAAACATAAAACAAGAAGGATATA 4740
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AATTTTACCCTGCATTTATTTTCTTAGTGACAAGGGTGATAAACTCAAAT 4790
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ACAGCTTTTAGAACTGGTTACAATAGCGACGGAGAGTTAGGTTATTGGGA 4840
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TAAGTTAGAGCCACTTTATACAATTTTTGATGGTGTATCTAAAACATTCT 4890
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTGGTATTTGGACTCCTGTAAAGAATGACTTCAAAGAGTTTTATGATTTA 4940
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TACCTTTCTGATGTAGAGAAATATAATGGTTCGGGGAAATTGTTTCCCAA 4990
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AACACCTATACCTGAAAATGCTTTTTCTCTTTCTATTATTCCATGGACTT 5040
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CATTTACTGGGTTTAACTTAAATATCAATAATAATAGTAATTACCTTCTA 5090
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCCATTATTACAGCAGGAAAATTCATTAATAAAGGTAATTCAATATATTT 5140
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ACCGCTATCTTTACAGGTACATCATTCTGTTTGTGATGGTTATCATGCAG 5190
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GATTGTTTATGAACTCTATTCAGGAATTGTCAGATAGGCCTAATGACTGG 5240
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTTTTATAATATGAGATAATGCCGACTGTACTTTTTACAGTCGGTTTTCT 5290
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AATGTCACTAACCTGCCCCGTTAGTTGAAGAAGGTTTTTATATTACAGCT 5340
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAG 5390
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGG 5440
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGC 5490
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCAT 5540
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTC 5590
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACATGGATGAGCGATGAT 5640
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GATATCCGTTTAGGCTGGGCGGTGATAGCTTCTCGTTCAGGCAGTACGCC 5690
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCTTTTCTTTTCCAGACCTGAGGGAGGCGGAAATGGTGTGAGGTTCCCGG 5740
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GGAAAAGCCAAATAGGCGATCGCGGGAGTGCTTTATTTGAAGATCAGGCT 5790
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ATCACTGCGGTCAATAGATTTCACAATGTGATGGCTGGACAGCCTGAGGA 5840
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ACTCTCGAACCCGAATGGAAACAACCAGATATTTATGAATCAGCGCGGCT 5890
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CACATGGCGTTGTGCTGGCAAATGCAGGTTCATCCTCTGTCTCTATCAAT 5940
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ACGGCAACAAAATTGCCTGATGGCAGGTATGACAATAAAGCTGGAGCGGG 5990
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTCATTTCAAGTGAACGATGGTAAACTGACAGGCACGATCAATGCCAGGT 6040
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTGTAGCTGTGCTTTATCCTGATGATATTGCAAAAGCGCCTCATGTTTTC 6090
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTTGAGAATTACAAAACAGGTGTAACACATTCTTTCAATGATCAACTGAC 6140
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GATTACCTTGCGTGCAGATGCGAATACAACAAAAGCCGTTTATCAAATCA 6190
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ATAATGGACCAGACGACAGGCGTTTAAGGATGGAGATCAATTCACAATCG 6240
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GAAAAGGAGATCCAATTTGGCAAAACATACACCATCATGTTAAAAGGAAC 6290
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GAACAGTGATGGTGTAACGAGGACCGAGAAATACAGTTTTGTTAAAAGAG 6340
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ATCCAGCGTCGGCCAAAACCATCGGCTATCAAAATCCGAATCATTGGAGC 6390
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CAGGTAAATGCTTATATCTATAAACATGATGGGAGCCGAGTAATTGAATT 6440
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GACCGGATCTTGGCCTGGAAAACCAATGACTAAAAATGCAGACGGAATTT 6490
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ACACGCTGACGCTGCCTGCGGACACGGATACAACCAACGCAAAAGTGATT 6540
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTTAATAATGGCAGCGCCCAAGTGCCCGGTCAGAATCAGCCTGGCTTTGA 6590
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTACGTGCTAAATGGTTTATATAATGACTCGGGCTTAAGCGGTTCTCTTC 6640
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCCATTGAGGGCAAGGCTAGACGGGACTTACCGAAAGAAACCATCAATGA 6690
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TGGTTTCTTTTTTGTTCATAAATCAGACAAAACTTTTCTCTTGCAAAAGT 6740
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTGTGAAGTGTTGCACAATATAAATGTGCGAAACGATCCTCATCCTGTCT 6790
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTTGATCCATGGATTACGCGTTAACCCGGGCCCGCGGATGCATATGATCA 6840
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GATCTTAAGGCCTAGGTCTAGAGGATCGATCTGTATAATAAAGAATAATT 6890
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ATTAATCTGTAGACAAATTGTGAAAGGATGTACTTAAACGCTAACGGTCA 6940
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCTTTATTGAACAGTAATTTAAGTATATGTCCAATCTAGGGTAAGTAAAT 6990
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TGAGTATCAATATAAACTTTATATGAACATAATCAACGAGGTGAAATCAT 7040
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GAGCAATTTGATTAACGGAAAAATACCAAATCAAGCGATTCAAACATTAA 7090
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AAATCGTAAAAGATTTATTTGGAAGTTCAATAGTTGGAGTATATCTATTT 7140
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GGTTCAGCAGTAAATGGTGGTTTACGCATTAACAGCGATGTAGATGTTCT 7190
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AGTCGTCGTGAATCATAGTTTACCTCAATTAACTCGAAAAAAACTAACAG 7240
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AAAGACTAATGACTATATCAGGAAAGATTGGAAATACGGATTCTGTTAGA 7290
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCACTTGAAGTTACGGTTATAAATAGGAGTGAAGTTGTCCCTTGGCAATA 7340
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCCTCCAAAAAGAGAATTTATATACGGTGAGTGGCTCAGGGGTGAATTTG 7390
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AGAATGGACAAATTCAGGAACCAAGCTATGATCCTGATTTGGCTATTGTT 7440
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TTAGCACAAGCAAGAAAGAATAGTATTTCTCTATTTGGTCCTGATTCTTC 7490
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AAGTATACTTGTCTCCGTACCTTTGACAGATATTCGAAGAGCAATTAAGG 7540
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ATTCTTTGCCAGAACTAATTGAGGGGATAAAAGGTGATGAGCGTAATGTA 7590
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ATTTTAACCCTAGCTCGAATGTGGCAAACAGTGACTACTGGTGAAATTAC 7640
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTCGAAAGATGTCGCTGCAGAATGGGCTATACCTCTTTTACCTAAAGAGC 7690
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ATGTAACTTTACTGGATATAGCTAGAAAAGGCTATCGGGGAGAGTGTGAT 7740
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GATAAGTGGGAAGGACTATATTCAAAGGTGAAAGCACTCGTTAAGTATAT 7790
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GAAAAATTCTATAGAAACTTCTCTCAATTAGGCTAATTTTATTGCAATAA 7840
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CAGGTGCTTACTTTTCTGGAGTTCTTTAGCAAATTTTTTTATTAGCTGAA 7890
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTTAGTATTAGTGGCCATACTCCTCCAATCCAAAGCTATTTAGAAAGATT 7940
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ACTATATCCTCAAACAGGCGGTAACCGGCCTCTTCATCGGGAATGCGCGC 7990
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GACCTTCAGCATCGCCGGCATGTCCCCCTGGCGGACGGGAAGTATCCAGC 8040
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCGAGGTCGGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTG 8090
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACA 8140
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTC 8190
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTT 8240
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG 8290
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCA 8340
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGG 8390
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGC 8440
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA 8490
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGC 8540
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CAGTTACCTTCGGAAAAAGAGTTGATAGCTCTTGATCCGGCAAACAAACC 8590
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG 8640
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG 8690
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCA 8740
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC 8790
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA 8840
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTT 8890
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATC 8940
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAG 8990
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGT 9040
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGC 9090
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTG 9140
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGC 9190
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica TCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAA 9240
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGG 9290
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACT 9340
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA 9390
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGT 9440
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATC 9490
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica ATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT 9540
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCAT 9590
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT 9640
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACT 9690
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA 9740
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCG 9790
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTAT 9840
Rat3 --------------------------------------------------
pJS150_seq_-_Ratiometrica CATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTC 9890
Rat3 -------
pJS150_seq_-_Ratiometrica AAGAATT 9897
Our ratiometric luciferase construct arrived with full sequence coverage. Unfortunately, it was unexpectedly toxic. This hampered characterisation, and necessitated its submission to the registry in a low copy number backbone.
TO ADD:
luminescence images orange, cosegregation
Instrumentation (Biologger)
Having our instrumentation completed, as can be seen in our Instrumentation (Biologger) page, the sensitivity of the sensor placed in the right position was tested using a dilution series of luciferase-producing E.coli. 20ml Cultures were grown overnight from single colonies. The cultures were induced with 40ul of 1.5M arabinose (for a final concentration of 3mM). Cultures were left for 2 1/2 hours for full induction. Subsequently, a culture was pelleted and resuspended in 4ml LB. Doubling dilutions, of volume 2ml, were made from this concentrate, down to 1/8th concentration. 1ml of each 2ml dilution was analysed in each cuvette, which was placed in the cuvette holder we made ourselves. The result was very good. An almost linear relationship was obtained when data were normalised with the sensor value taken in the dark room (the latter set at zero) without using the cuvette holder (1-(sensor value/sensor value in absolute dark)), presenting the sensitivity of the sensor to different intensities of light. This behaviour was expected due to the changing offset affecting the luciferase spectrum curve at different light intensities. The offset, using our data, was calculated to be about 0.2V for each dilution. A second graph is shown which takes into account this offset (and removes it), thus showing the presence of blue frequencies. The result was as expected, as the presence of blue frequencies throughout the dilution series is not only detected, but also found to be approximately constant. The raw data of this investigation can be found in the Lab book.
Once the sensor was tested for sensitivity, we tested that our circuitry correctly identified different frequencies (colours) of light. As can be seen below, measurements taken from orange and blue light yield values respectively above and below those from white light (our reference point). The data was taken using constant intensity of light for each case (V.High and V.Low brightness, as specified in the application). This was done with the aid of an Android phone and a specialised software application, called Color Flashlight, downloaded from the official Market.
As expected from the potential-divider design of our circuitry, orange and red frequencies caused the resistance of the LDR with the orange filter to decrease, leading to a higher voltage across the LDR with the blue filter. The opposite effect was observed with blue light. The reason that the white reference point is a bit lower than 2.5V (the expected value for a non-biased circuitry with a 5V source), is because we use resistors of total net resistance 1.67 kΩ before the blue LDR. This was done to bias the circuitry towards blue (i.e. decreasing the starting value, thus the sensor identifies always a bit more blue - this can be shown in previous graphs as well) and thus cause orange light to have a larger impact when present. This was used in an attempt to compensate for the fact that the peak at 560nm (Orange) in MOrange/luciferase fusion spectrum is lower than the one at 490 nm (Blue). Even though we did not manage to test the latter with transformed bacteria, the data collected in all the previous experiments makes us confident that the instrumentation is at least adequately functional.
As the major part of the instrumentation, the bio-electronic interface, had been made and tested, now we turned to testing the other parts of our deveoped kit. This included the mechanical chassis of the prototype, the electronic/mechatronic (sensory and motory) components, and of course the software. The overview of the finished hardware/software can be seen in our Intstrumentation (Biologger) page. Below, the videos showing our instrumentation in action can be seen.
