Results

                       A                                            B

Fig. 1 – A: Double RE digest (EcoRI and PstI) of pBAD-Bovine Galectin 1 and OmpA-Bovine Gibson assemblies. For pBAD-BtGal1, all lanes for after ladder have bands of the correct weight, while no lanes for OmpA-Bovine are of the correct weight. B: Expected band sizes after double digest for pBAD-Bovine Galectin 1. The EcoRI cut site in the Bovine Galectin 1 gene was subsequently removed via site directed mutagenesis.

Mammalian galectins typically have a simpler quaternary structure than invertebrate galectins; they are usually dimers while invertebrate galectins are usually tetramers. Bovine galectin 1, from Bos taurus, is a simpler galectin than CvGal1 and can thus be utilized as a model galectin to anchor in the E. coli outer membrane. In addition, while the crystal structure of CvGal1 and the exact binding ligand on P. marinus that CvGal1 binds to are still under investigation, the crystal structure and nature of bovine galectin 1 have been well studied. We have cloned Bovine galectin 1 into the pSB1C3 backbone via Gibson assembly. The bovine galectin 1 gene was ordered as a gBlock from IDT and PCR amplified for Gibson assembly. All Gibson products were verified via sequencing.

AB

 (3rd Lane contains OmpA-Bovine product)

Fig. 2 - OmpA-Bovine (cut with BamHI and HindIII, before SDM)

Fig. 2 – Double digest of OmpA-Bovine Galectin 1 (BamHI and HindIII) Lane 3 contains bands of the correct weight after digest B: Expected band sizes after double digest for pBAD-Bovine Galectin 1. Site directed mutagenesis was subsequently performed in order to remove an EcoRI site in the Bovine galectin 1 gene. Figure 2A shows the Bovine galectin-1 assembled into pSB1C3-pBAD-RBS as a fusion protein with OmpA. When expressed in E. coli, the galectin should continue off the C-terminus of the OmpA-linker and thus be transported to the extracellular matrix since it is attached to the OmpA transport protein. After plasmid assembly transfer of plasmid into a DH5α E. coli cells strain, the plasmid was extracted and digested with EcoRI and PstI. The pattern of three bands appears again due to an additional EcoRI restriction site.

Sequencing Results:

OmpA-Bovine SDM Chromatogram:
Sequence file:
NNNNNNNNNNNTNNNNANNNNNAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATG
ATTTCTGGAATTCGCGGCCGCTTCTAGAGACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCAAGATAGT
CCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCGTTTTTTTGGGCTAGCT
ACTAGAGAAAGAGGAGAAATACTAGCATATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGC
TGGCAGGTTGCTCCAGCAACGCTAAAATCGATCAGGGAATTAACCCGTATGTTGGCTTTGAAATGGGTTACGACTGGTTA
GGTCGTATGCCGTACAAAGGCAGCGTTGAAAACGGTGCATACAAAGCTCAGGGCGTTCAACTGACCGCTAAACTGGGTTA
CCCAATCACTGACGACCTGGACATCTACACTCGTCTGGGTGGCATGGTATGGCGTGCAGACACTAAATCCAACGTTTATG
GTAAAAACCACGACACCGGCGTTTCTCCGGTCTTCGCTGGCGGTGTTGAGTACGCGATCACTCCTGAAATCGCTACCCGT
CTGGAATACCAGTGGACCAACAACATCGGTGACGCACACACCATCGGCACTCGTCCGGACAACGGCGGANGTTCTGGAGG
ANGGAGCATGGCGTCGGGGCTTGTTGCGTCCAATCTGAATCTGAAACCAGGTGAAAGCTTACGCGTCCGCGGCGAAGTAG
CCGCAGATGCGAAATCTTTTCTCTTGAACCTGGGCAAGGACGANAATAATCTGGCTTTGCATTTCAACCCGCGTTTCNAC
GCTCATGGNGACNTGAACACAATCNTGTGTAANTCCAAGGNTGCTGGTGCTTGGGGTGCANAANNANCGNGNNNTCCGCG
TTCCCGTTTCANNNNNGCTCANNNNGGNAGTCACCNTNCNGCNTTCANCCANNNCGANNTNCNNNNTTAAANNNGNNNNN
NGGNNNCNAAGTTNNANTTNNCNAANCNNNTNNAACNNNNNGNNNNNNNNNNNNNNNNNGNNNN

A

pBAD-Bovine SDM chromatogram:
NNNNNNNNNNNNTNNNCTATNNNNTAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGG
ATGATTTCTGGAATTCGCGGCCGCTTCTAGAGACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCAAGAT
AGTCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCGTTTTTTTGGGCTA
GCTACTAGAGAAAGAGGAGAAATACTAGATGGCGTCGGGGCTTGTTGCGTCCAATCTGAATCTGAAACCAGGTGAAAGCT
TACGCGTCCGCGGCGAAGTAGCCGCAGATGCGAAATCTTTTCTCTTGAACCTGGGCAAGGACGATAATAATCTGGCTTTG
CATTTCAACCCGCGTTTCAACGCTCATGGCGACGTGAACACAATCGTGTGTAATTCCAAGGATGCTGGTGCTTGGGGTGC
AGAACAGCGTGAGTCCGCGTTCCCGTTTCAACCGGGCTCAGTTGTGGAAGTCACCATCAGCTTCAACCAGACCGATCTCA
CCATTAAACTGCCGGATGGCTACGAGTTTAAGTTTCCAAACCGTTTAAACCTCGAGGCCATCAACTATCTGTCCGCGGGG
GGCGACTTCAAGATTAAAAGCGTGCACCATCATCACCACCATTGATCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCG
TTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGA
AAAGATTACTTCGCGTTATGCNGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCA
GCTCACTCAAAGGCGGTAATACGGTTATCNNCAGAATCNGGGGATNACGCAGGAAANAACATGTGAGCAAANNNCAGCAA
AANGCAGGAANCGTAAAANNNN

B

Fig. 3 - Sequencing results and Chromatogram of SDM’d plasmids (BBa_K1489000 and BBa_K1489004)

To address this issue, site-directed mutagenesis was performed on these plasmids to change the EcoRI sequence within the bovine galectin-1 gene (5’-GAATTC-3’) to 5’-GAGTTT-3’, which will code for the same Glu-Phe sequence. Figure 3A and 3B demonstrate the two successfully mutagenized base pairs in the bovine galectin-1 gene, while preserving the rest of the plasmid.

 Sequencing results: NNNNNNNNNNNNNNNNTATANAAATAGGCGTATCNCGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGG ATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGCATATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTT CTACTCTGCTGGCAGGTTGCTCCAGCAACGCTAAAATCGATCAGGGAATTAACCCGTATGTTGGCTTTGAAATGGGTTAC GACTGGTTAGGTCGTATGCCGTACAAAGGCAGCGTTGAAAACGGTGCATACAAAGCTCAGGGCGTTCAACTGACCGCTAA ACTGGGTTACCCAATCACTGACGACCTGGACATCTACACTCGTCTGGGTGGCATGGTATGGCGTGCAGACACTAAATCCA ACGTTTATGGTAAAAACCACGACACCGGCGTTTCTCCGGTCTTCGCTGGCGGTGTTGAGTACGCGATCACTCCTGAAATC GCTACCCGTCTGGAATACCAGTGGACCAACAACATCGGTGACGCACACACCATCGGCACTCGTCCGGACAACGGCGGAGG TTCTGGAGGAGGGAGCTCTACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTT CTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGG CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCANGNNNAACATGTGAGCA AAAGGCCAGCNNAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCAT CACAAAAATCGACGCTCAAGTCAGANGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGNAGCTC CCTCGTGCGCTCTCCTGTTCCNACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCNTAGCTCACGCTGTAGGTATCNNCANNN

Fig 4. - Sequencing Results and Chromatogram of OmpA moved into pSB1C3

Since UMaryland worked extensively with the OmpA-linker (BBa_K103006), we have also looked into improvements on that BioBrick.

OmpA-KasI chromatogram, shrunk down

Sequencing Results:

Sequence:
NNNNNNNNNNNNNNNTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGA
TGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGCATATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTC
TACTCTGCTGGCAGGTTGCTCCAGCAACGCTAAAATCGATCAGGGAATTAACCCGTATGTTGGCTTTGAAATGGGTTACG
ACTGGTTAGGTCGTATGCCGTACAAAGGCAGCGTTGAAAACGGTGCATACAAAGCTCAGGGCGTTCAACTGACCGCTAAA
CTGGGTTACCCAATCACTGACGACCTGGACATCTACACTCGTCTGGGTGGCATGGTATGGCGTGCAGACACTAAATCCAA
CGTTTATGGTAAAAACCACGACACCGGCGTTTCTCCGGTCTTCGCTGGCGGTGTTGAGTACGCGATCACTCCTGAAATCG
CTACCCGTCTGGAATACCAGTGGACCAACAACATCGGTGACGCACACACCATCGGCACTCGTCCGGACAACGGCGGAGGT
TCTGGAGGAGGGGCGCCTACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTC
TTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGC
GAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCA
AAAGGNCAGCAAAAGGNCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCA
TCACAAAAATCGACGCTCAAGTCANNNNGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTC
CCTCGTGCGCTCTNCTGTTCCNACCCTGCCGCTTACCNGATACCTGTCNCNTTCTNCCTTCGGGANCGNGNGCTTTCTCA
TAGCTCACGCTNTAGGTANNNCANTNCGGNNNTNGNCGTTNNNCNCNANNCTNGNNTN

Fig. 5 - Sequencing Results and Chromatogram of SDM’d OmpA in pSB1C3

The previous version of Lpp-OmpA-Linker (BBa_K103006), designed for the pSB1A2 vector backbone, contained the non-traditional restriction sites NdeI (CATATG) and SacI (GAGCTC) in order to attach proteins at the N-terminus and C-terminus of OmpA, respectively. While the NdeI site is still useful for RE cloning at the N-terminus of OmpA, the implementation of the new plasmid backbone pSB1C3 has made the SacI site difficult to use. This is due to the presence of a second SacI site in the chloramphenicol acetyltransferase gene in pSB1C3. We have thus decided to mutagenize the previous SacI site on Lpp-OmpA-Linker into a KasI site in order to remove this issue. The KasI site (GGCGCC) encodes the amino acids glycine and alanine, converting the sequence of the linker from Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser to Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ala. We believe this to be a sufficiently minor alteration to the unfolded linker sequence as to not affect protein function.

Fig. 6 - SWestern Blot of expression of SDM’d OmpA-BovineGalectin in pSB1C3

"Fig. 6 - Expression test for pBAD-Bovine galectin 1 and OmpA-Bovine galectin 1. Western Blot with anti-His antibody was done on soluble and insoluble fractions of non-induced cells and induced cells (0.2% arabinose for 21 hours). Bands can be observed in induced cells for pBAD-Bovine galectin 1 and both induced and non-induced cells for OmpA-Bovine galectin 1"