1. Measuring fluorescence of GFP
Yesterday, varieties of bacterial cultures were grown in order to test the expression of GFP's. The fluorescence of GFP are measured by the plate reader, and following are the results that Emily and I were able to get today:
- Fluorescent levels of different cultures (Stored in the plate reader for 5 hours after dilution of R0040+GFP)
- Fluorescent levels of different cultures (Stored in the shaker at 37˚C for 5 hours after dilution of R0040+GFP)
We were forced to wait for 5 hours after the dilution of R0040+GFP culture as we were asked to leave the lab that had the plate reader by another organization. We were able to measure once quickly right after the dilution; however, with no accurate settings, meaning we cannot make any comparisons.
As for the above results, it seems convincing enough for me to say those cells that have the reporter and mutant (LuxOD47E) circuits in them are doing something special that make their readings higher. Our KT1144 cells have a cosmid that contains Qrr4 promoter driving an expression of GFP; thus, as shown by the data, express GFP in larger quantities when it has the LuxOD47E mutant than when it doesn't. Our TOP10 cells also have a plasmid containing Pqrr4 and GFP, which behaves the same way as our KT1144 cells.
Both data show that the fluorescence reading is about 2 times higher in cells that have both mutants and reporters than the cells that doesn't have mutant circuits. As we can see in well B7, the cells that have R0040, a constitutive promoter, driving GFP have a much higher reading than the rest, which is what was expected. Also as expected, diluting the R0040+GFP cells to 1/10, 1/50, and 1/100 ratios resulted in much lower readings of fluorescence. Two interesting things to note about the above collected data are:
1. Both with and without mutant readings of TOP10 cells with reporter are than the readings of KT1144 cells. I do not know exactly why it is as such, but my guess is that different type of cells absorb different amount of wavelengths.
2. The fluorescent levels of the cells that was grown for an extra 5 hours in the shaker were lower than the cells that grew in the plate reader, except for the cells that were diluted with fresh LB media. As above, I do not know the exact reason. Perhaps it is because the cells have already reached their 16~20 hours of growing overnight, growing for an extra 5 more hours at 37˚C killed the bacteria, decreasing the production of GFP, while the growth of the cells that were stored in the plate reader for an extra 5 hours slowed down due to a relatively colder temperature, thus being able to produce GFP more stably.
As for the diluted cells with R0040+GFP, because they were transferred to a fresh media before the additional growth of 5 hours, storing them in the shaker at 37˚C led to faster replication, which sped up the production of GFP, while storing them in the colder plate reader slowed down the replication, which slowed the production of GFP.
2. Sequencing results
The sequencing results of
Pqrr4+B0034+K082003 came back today. Unfortunately, B0034 was still not there despite the change of
Pqrr4+B0034 colonies from C9 to C1. Surprisingly, the sequence for the previous construction that used
Pqrr4+B0034 C9 actually match the sequence that came back today, which used
Pqrr4+B0034 C1. (99% match) The following is the alignment of the two sequences:
>lcl|28877
Length=801
Score = 1472 bits (797), Expect = 0.0
Identities = 800/801 (99%), Gaps = 1/801 (0%)
Strand=Plus/Plus
Query 9 GGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATTAAGCTACTAA 68
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1 GGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATTAAGCTACTAA 60
Query 69 AGCGTAGTTTTCGTCGTTTGCAGCAGGCCTTTTGTATAGTTCATCCATGCCATGTGTAAT 128
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 61 AGCGTAGTTTTCGTCGTTTGCAGCAGGCCTTTTGTATAGTTCATCCATGCCATGTGTAAT 120
Query 129 CCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTT 188
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 121 CCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTT 180
Query 189 CGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCC 248
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 181 CGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCC 240
Query 249 AATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTG 308
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 241 AATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTG 300
Query 309 TCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATT 368
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 301 TCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATT 360
Query 369 GTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATC 428
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 361 GTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATC 420
Query 429 AATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACG 488
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 421 AATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACG 480
Query 489 TGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGG 548
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 481 TGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGG 540
Query 549 CATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTG 608
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 541 CATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTG 600
Query 609 AACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGT 668
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 601 AACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGT 660
Query 669 GCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGAC 728
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 661 GCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGAC 720
Query 729 AGAAAA-TTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAG 787
|||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 721 AGAAAAATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAG 780
Query 788 TGAAAAGTTCTTCTCCTTTAC 808
|||||||||||||||||||||
Sbjct 781 TGAAAAGTTCTTCTCCTTTAC 801
And the following is the previous sequencing result that I have gotten for Pqrr4+B0034 that was used for construction:
Sequence of Pqrr4+B0034 (RBS) C1
Legend
Vector contamination
BBK Restriction sites
Pqrr4 Sequence
B0034 sequence
GGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTG
GAATTCGCGGCCGCTTCTAGAGTATCAGCAAAAACACTACGGTGGATAATCAGTAAAACCATGAAACTAGAGCCCCGCA
CACTTGCGGGGCTTTTTAATTTTGAATTTCTTTCTTATTAAAACGCCATTTTTCTGATAAATGTATTAGTAGCAA
TGCGCATGGTGGCATATTTGCATCATTTTGCATTTTGCAAATGCGATTTGCAAAATGCGTGCTCAATAAAGCACC
AATATGCATCAGGATCGAAGAAAAAAGGCGTTTTTAAAAGTTGGCACGCATCGTGCTTTATACAGATACTAGAGAAAGAGGAGAAA
TACTAGTAGCGGCCGCTGCAG
TCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTT
CGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGG
CGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCG
TAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG
TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC
CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC
GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT
CGTCTTGAGTCCAACCCGGTAGACACGACTTATCGCCACTG
When I carefully analyzed the sequence, I could find part of the B0034 embeded between Pqrr4 and K082003. The following explains this further:
Legend
end of Pqrr4 sequence
Biobrick restriction sites
Part of B0034 (Complete sequence is AAAGAGGAGAAA
Part of scar? (Complete sequence is ACTAGAG)
Beginning of K082003
...AGATACTAGAGAAAGAGGCTAGATGCGTA...
A mistake of sending two wrong colonies of Pqrr4+B0034 for sequencing is highly unlikely, and also a mistake of using a wrong colony for construction two times is highly unlikely, especially because I was not willing to mess up the construction the second time. I will have to investigate this further.
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