Team:UMaryland/project/results

From 2014.igem.org

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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>
<p><strong style="font-weight: normal;"> </strong></p>
<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;">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>

Revision as of 19:25, 17 October 2014

Results

About Umaryland

UMaryland2014 is University of Maryland, College Parks, inaugural iGEM team. We are a combined effort of several departments and numerous faculty mentors. Although it is only our first year, believe our hard work and dedication has paid off. We can't wait for this years competition! GO TERPS!