Team:UMaryland/project/results

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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><img src="https://static.igem.org/mediawiki/2014/e/e0/UMFig1-1.jpg" border="0" width="213px;" height="248px;" style="border: NaNpx solid black; transform: rotate(0.00rad); -webkit-transform: rotate(0.00rad);" /></span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><img src="https://static.igem.org/mediawiki/2014/2/28/UMFig1-2.png" border="0" width="354px;" height="148px;" style="border: NaNpx solid black; transform: rotate(0.00rad); -webkit-transform: rotate(0.00rad);" /></span></p>
<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><img src="https://static.igem.org/mediawiki/2014/e/e0/UMFig1-1.jpg" border="0" width="213px;" height="248px;" style="border: NaNpx solid black; transform: rotate(0.00rad); -webkit-transform: rotate(0.00rad);" /></span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><img src="https://static.igem.org/mediawiki/2014/2/28/UMFig1-2.png" border="0" width="354px;" height="148px;" style="border: NaNpx solid black; transform: rotate(0.00rad); -webkit-transform: rotate(0.00rad);" /></span></p>
<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">                        A                                            B</span></p>
<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">                        A                                            B</span></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Fig. 1 - Bovine Galectin-1 gene cloned into the pSB1C3 vector with pBAD promoter and a RBS</span></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">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.</span></p>
<p><strong id="docs-internal-guid-809a97bc-f600-9f66-fada-73accace1d98" style="font-weight: normal;"> </strong></p>
<p><strong id="docs-internal-guid-809a97bc-f600-9f66-fada-73accace1d98" style="font-weight: normal;"> </strong></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Galectin-1 from </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: italic; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Bos taurus</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">  has been cloned into pSB1C3 vector. </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: italic; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Bos taurus </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">or bovine galectin enabled us to generate a platform that enables future work on ligand binding. While CvGals binding characteristics are still being researched the crystal structure and nature of bovine galectin has been well studied. The subsequent BioBricks regarding this gene were all constructed via Gibson assembly and cloning. The plasmids from which the OmpA insert and the vector were amplified via extension PCR are respectively: OmpA with linker (BBa_K103006) and pBAD-RBS on the pSB1C3 backbone (BBa_K750000). For the latter, the GFP in the BioBrick was not included for Gibson assembly. The bovine galectin-1 was ordered as a synthetically synthesized sequence from IDT as a linear gene and amplified directly with appropriate primers for Gibson assembly. </span></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">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. </span></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Figure </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: bold; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">1A</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> shows the Bovine galectin-1 assembled into pSB1C3-pBAD-RBS alone (no OmpA), which when expressed in </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: italic; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">E. coli</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> should produce a soluble form of the protein. After successful assembly and cloned into DH5α </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: italic; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">E. coli</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">. The plasmid was cut with restriction enzymes EcoRI and PstI, standard sites in the pSB1C3 vector’s BioBrick prefix and suffix, respectively. The result is expected to yield the insert, pBAD-RBS-Bovine galectin-1, and the pSB1C3 vector. However, an unwanted EcoRI site was found in the galectin-1 gene afterwards, yielding the pattern seen in the gel. Figure </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: bold; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">1B</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> demonstrates the expected pattern, with band sizes of 2033 bp, 496 bp, and 169 bp top to bottom, via the software “A plasmid Editor” (ApE, courtesy of M. Wayne Davis, University of Utah).</span></p>
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<p><strong style="font-weight: normal;"> </strong></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><img src="https://static.igem.org/mediawiki/2014/3/3f/UMFig2-1.jpg" border="0" width="233" height="331" style="border: 0;" />A<img src="https://static.igem.org/mediawiki/2014/e/ef/UMFig2-2.png" border="0" width="354px;" height="151px;" style="border: 0; transform: rotate(0.00rad); -webkit-transform: rotate(0.00rad);" /></span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">B</span></p>
<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><img src="https://static.igem.org/mediawiki/2014/3/3f/UMFig2-1.jpg" border="0" width="233" height="331" style="border: 0;" />A<img src="https://static.igem.org/mediawiki/2014/e/ef/UMFig2-2.png" border="0" width="354px;" height="151px;" style="border: 0; transform: rotate(0.00rad); -webkit-transform: rotate(0.00rad);" /></span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">B</span></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Fig. 2 - </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: bold; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">OmpA-Bovine (cut with BamHI and HindIII, before SDM)</span></p>
<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Fig. 2 - </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: bold; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">OmpA-Bovine (cut with BamHI and HindIII, before SDM)</span></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Figure </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: bold; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">2A</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> shows the Bovine galectin-1 assembled into pSB1C3-pBAD-RBS as a fusion protein with OmpA. When expressed in </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: italic; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">E. coli</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">, 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 </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: italic; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">E. coli</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> 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. </span></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">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 </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: bold; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">2A</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> shows the Bovine galectin-1 assembled into pSB1C3-pBAD-RBS as a fusion protein with OmpA. When expressed in </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: italic; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">E. coli</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">, 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 </span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: italic; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">E. coli</span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> 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. </span></p>
<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> </span></p>
<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> </span></p>
<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> </span></p>
<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"> </span></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Fig. 6 - <strong>S</strong></span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: bold; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Western Blot of expression of SDM’d OmpA-BovineGalectin in pSB1C3</span></p>
<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: normal; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Fig. 6 - <strong>S</strong></span><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: bold; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Western Blot of expression of SDM’d OmpA-BovineGalectin in pSB1C3</span></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: bold; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><br /></span><span style="line-height: 12.777777671814px;">"Both the bovine galectin and the OmpA-Bovine Galectin were tested for expression using a Western blot. Each sample was also tested with and without induction. Additionally for all trials both the soluable and insoluable fractions were examined. The results showed expression of both the Bovine galectin and the OmpA-Bovine galectin. The conclusion points to sucessful expression of our biobrick "</span></p>
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<p style="line-height: 1.15; margin-top: 0pt; margin-bottom: 0pt;" dir="ltr"><span style="font-size: 15px; font-family: Arial; color: #000000; background-color: transparent; font-weight: bold; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><br /></span><span style="line-height: 12.777777671814px;">"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"</span></p>
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Latest revision as of 19:42, 17 October 2014

Results

About Umaryland

UMaryland 2014 is the inaugural iGEM team of the University of Maryland, College Park. We are a combined effort of several departments and numerous faculty mentors. Although it is only our first year, we believe our hard work and dedication has paid off. We can't wait for this year's competition! GO TERPS!