PCR and sequencing work

Objective: Since the sequencing data that was collected did not fit the luciferase sequencing obtained from Renilla reniformes, we are running another. Started a PCR  using all the measurements from 4/22/2015 using only the luciferase primers. Through Further research I did  find multiple studies involving the bacteria and luciferase, though I am not sure if that has a connection to the results

Liu, Y., Abaibou, H., & Fletcher, H. M. (2000). Development of a noninvasive reporter system for gene expression in Porphyromonas gingivalis. Plasmid,44(3), 250-261.

Coats, S. R., Reife, R. A., Bainbridge, B. W., Pham, T. T. T., & Darveau, R. P. (2003). Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at toll-like receptor 4 in human endothelial cells.Infection and immunity, 71(12), 6799-6807.

1 RR Luciferase
2 RR Luciferase
3 H20 Control
4 H20 Control
Dr. Roberts suggested blasting each sequence


Results: Sucessfully prepared a gel and started the PCR. Spent the remaining time working with Sam to go over Sequencing data.

Geneious work

Worked with sam to go over sequencing data using geneious 5.3.6 trimmed all 12 sequences, aligned and then assembled shown below. The red areas are the areas that were trimmed leaving the un-red areas to be aligned. Blasted the sequence that was created in NCBI using nucleotide blast, tbalstx (compares proteins), and Compared directly to the sequence that was used to prepare primers. Both blasts did not return anything related to Luciferase or Renilla Reniformes. Since this sequence has never been sequenced before may be why nothing is relating. The next PCR is completely focused on luciferase which gave us good results last run through. I am planning on going through and blasting each sequence at a later date. Sam and I both think there may be something going on since the sequencing results are a lot shorter than what was being sent out. The PCR bands were over 800 base pairs but all the sequencing data has been between 200 to 300 bp.

Residues:
(-------------------------------CGGCTCTGCCTCGCGTATCGATAT-GATTTGGTTGCCTACCCGCTTTTCCGGGTTTATTAATAAAGTWRAAATCTTACCATGTTTCCGGTCGGACCATCATTATGTGTTTATTGAGATGCATTTACCTTTTTCTGTTGTTCGCGGCAATGAGCTTTGGAAACTCAATTTTTCATTATTAAAAGACGAATGTTTATGCCAGAAAATTATGGACTTCTGGAAGATATGGAAGGTCCAAAAACATGTTTACCTTCCCTCCGTTTGGTGGGAACTTGGAAAAAAGCGTCTTATCGACATTATCCGCCGTTTCGGTCGAAGCCKTGCTAGCGCAGTGCGCGATCGTGTTGCTGATTTAACTGCCCAATTGAATGCATGCAAAA-----------------)

Went into the literature to reverify the presence of luciferase in ptilosarcus gurneyi did not find much information  only one study (shown below)

Shimomura, O., & Johnson, F. H. (1979). Comparison of the amounts of key components in the bioluminescence systems of various coelenterates.Comparative Biochemistry and Physiology Part B: Comparative Biochemistry,64(1), 105-107.