Team:Aberdeen Scotland/Parts/ 2006

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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li>
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li>
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li>
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li>
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li>
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li>
<li class="curr"><a class="curr" href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2006">Bba_K1352006</a></li>
<li class="curr"><a class="curr" href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2006">Bba_K1352006</a></li>
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<div class="t_overview">
<div class="t_overview">
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<h1>Background to Parts Design</h1>
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<h1><br>INP+FLAG</h1>
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K1352006 is a part which has a FLAG-tag octapeptide flanked by a multiple cloning site (MCS) inserted into Bba_K523013 (K523013 expresses ice nucleation protein (INP)); specifically, on the end of a linker, which is in turn on the C-terminus of INP and is in the same reading frame. The FLAG-tag is a BglII restriction site, followed by a FLAG octapeptide, followed by a HindIII restriction site; the sequence of which is as follows; the octapeptide is uppercase, the restriction sites are lowercase:<br>
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(agatctGATTATAAAGATGATGATGATAAAaagctt)<br>
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Attaching a FLAG-tag to the end of INP means that it will be expressed on the surface of the cell, the purpose of this is to allow the rapid insertion (via the MCS) of polypeptides (an antigen for example) to the C-terminus end of INP; this allows the surface expression of said polypeptide. For the 2014 Aberdeen iGEM project, this part was intended to be used as a trypanosome antigen displayer.<br><br>
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<h3>Creation of INP-YFP-FLAG fragments followed by InFusion cloning</h3>
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Four infusion primers (primers with one homologous half (lower case) to the template, and one “overhang” half (upper case), the last 15 nucleotides of which is homologous to another DNA fragment) were designed.<br><br>
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“INP-VEC-F” (CGCGGCCGCTTCTAGAttaatacgactcactataggg)<br>
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“INP-Rem-R” (aagctttttatcatcatcatctttataatcagatctTCCCGCCACGCTGC)<br>
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“INP-FLAG-F” (AGATCTGATTATAAAGATGATGATGATAAAAAGCTTtaataatactagcaacatatcataacggagtg)<br>
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“INP-VEC-R” (AGCGGCCGCTACTAGTtataaacgcagaaaggccc)<br><br>
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Two INP-YFP-FLAG fragments were created by PCR amplification, both use K523013 as the template, the first uses “INP-VEC-F” and “INP-FLAG-R” primers, the second uses “INP-VEC-R” and “INP-FLAG-F” primers. The Clontech InFusion kit was used to recombine the two INP-YFP-FLAG fragments with pSB1C3 backbone (cut with Xba1 and Spe1); this kit was followed according to the manufacturer’s instructions.<br><br>
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<h3>Restriction digest + Gel electrophoresis</h3><br>
<center>
<center>
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<img src="<center>
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<img src="https://static.igem.org/mediawiki/parts/1/18/2006_1.PNG">
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<img src="https://static.igem.org/mediawiki/2014/5/55/2002fig2.png">
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<br>
<br>
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<font size="2">Figure 1; a Xba1 + HindIII restriction digest screen of recombinants. The recombinant which went on to become K1352006 is in lane 5. The “L” lane is DNA marker (ladder). The arrows are to highlight the distance travelled by the HindIII-negative recombinants.
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<font size="2">Figure 1; Xba1 + HindIII restriction digest screen of recombinants. The recombinant which went on to become K1352006 is in lane 5. The “L” lane is DNA marker (ladder). The arrows are to highlight the distance travelled by the HindIII-negative recombinants.
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</font>
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<center>
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<img src="https://static.igem.org/mediawiki/parts/5/58/2006_2.PNG">
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<br>
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<font size="2">Figure 2; restriction digest verification of plasmid K1352006
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<br>LEGEND:<br>
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L 10,000bp – 500bp DNA marker “ladder”<br>
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N K1352006 plasmid digested with no enzymes<br>
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E K1352006 plasmid digested with EcoRI<br>
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X K1352006 plasmid digested with XbaI<br>
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S K1352006 plasmid digested with SpeI<br>
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P K1352006 plasmid digested with PstI<br>
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EP K1352006 plasmid digested with EcoRI and PstI<br>
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XS K1352006 plasmid digested with XbaI and Spe1<br>
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<br><br>">
 
<br>
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<font size="2">Fig.1&nbsp;&nbsp;&nbsp;&nbsp;Graphic representation of Ag43 autotransporter structure and process of autotransportation.
 
<br>
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<h3>DNA Sequencing</h3>
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The recombinant plasmid was Sanger-sequenced with the following sequencing primers. “G101” is a reverse primer, the rest are forward primers.<br><br>
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“G101” (attaccgcctttgagtgagc)<br>
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“G100” (tgccacctgacgtctaagaa)<br>
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“35 INP-SEQ 1” (ccgattcattaatgcagctgg)<br>
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“36 INP-SEQ 2” (gaggttgctgttgccgac)<br>
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“37 INP-SEQ 3” (ggtgtggaagccgacattc)<br><br>
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The plasmid insert was found to be exactly as desired. Highlighted below is the MCS excerpt from the “G101” primer results (“G101” is a reverse primer which is why the sequence is in reverse complement).<br><br>
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<center>
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<img src="https://static.igem.org/mediawiki/parts/0/06/2006_3.PNG">
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<br><br>
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<p>Antigen 43 (Ag43), the product of the </i>flu</i> gene, is a cell-surface autotransporter protein found in <i>Escherichia coli</i>. It is expressed at about 50,
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000 copies/cell and is initially synthesised as a precursor of 1039 amino acids. Upon removal of the signal peptide, the protein is transported to the cell surface
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and is composed of an α subunit (499 amino acids) at the N-terminus and a β subunit (488 amino acids) at the C-terminus. Ag43 is mainly known to induce cell-to-cell
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<h3>Conclusions</h3>
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aggregation and be involved in biofilm formation. However, as the necessary information required for auto transportation resides in the protein itself, the main of
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The part is RFC 10 compatible. The construct sequence was produced exactly as designed; the full FLAG-tag – with BglII and HindIII sites – is present at the N-terminus of the linker sequence after INP.<br><br>
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our project was to use it as a platform for displaying specific peptides on the surface of <i>E. coli</i>.</p>
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<img src="https://static.igem.org/mediawiki/2014/2/2e/Ag43.jpg" alt="Ag43">
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Latest revision as of 02:10, 18 October 2014

Team:Aberdeen Scotland/Parts - 2014.ogem.org




INP+FLAG


K1352006 is a part which has a FLAG-tag octapeptide flanked by a multiple cloning site (MCS) inserted into Bba_K523013 (K523013 expresses ice nucleation protein (INP)); specifically, on the end of a linker, which is in turn on the C-terminus of INP and is in the same reading frame. The FLAG-tag is a BglII restriction site, followed by a FLAG octapeptide, followed by a HindIII restriction site; the sequence of which is as follows; the octapeptide is uppercase, the restriction sites are lowercase:
(agatctGATTATAAAGATGATGATGATAAAaagctt)
Attaching a FLAG-tag to the end of INP means that it will be expressed on the surface of the cell, the purpose of this is to allow the rapid insertion (via the MCS) of polypeptides (an antigen for example) to the C-terminus end of INP; this allows the surface expression of said polypeptide. For the 2014 Aberdeen iGEM project, this part was intended to be used as a trypanosome antigen displayer.

Creation of INP-YFP-FLAG fragments followed by InFusion cloning

Four infusion primers (primers with one homologous half (lower case) to the template, and one “overhang” half (upper case), the last 15 nucleotides of which is homologous to another DNA fragment) were designed.

“INP-VEC-F” (CGCGGCCGCTTCTAGAttaatacgactcactataggg)
“INP-Rem-R” (aagctttttatcatcatcatctttataatcagatctTCCCGCCACGCTGC)
“INP-FLAG-F” (AGATCTGATTATAAAGATGATGATGATAAAAAGCTTtaataatactagcaacatatcataacggagtg)
“INP-VEC-R” (AGCGGCCGCTACTAGTtataaacgcagaaaggccc)

Two INP-YFP-FLAG fragments were created by PCR amplification, both use K523013 as the template, the first uses “INP-VEC-F” and “INP-FLAG-R” primers, the second uses “INP-VEC-R” and “INP-FLAG-F” primers. The Clontech InFusion kit was used to recombine the two INP-YFP-FLAG fragments with pSB1C3 backbone (cut with Xba1 and Spe1); this kit was followed according to the manufacturer’s instructions.

Restriction digest + Gel electrophoresis



Figure 1; Xba1 + HindIII restriction digest screen of recombinants. The recombinant which went on to become K1352006 is in lane 5. The “L” lane is DNA marker (ladder). The arrows are to highlight the distance travelled by the HindIII-negative recombinants.



Figure 2; restriction digest verification of plasmid K1352006
LEGEND:
L 10,000bp – 500bp DNA marker “ladder”
N K1352006 plasmid digested with no enzymes
E K1352006 plasmid digested with EcoRI
X K1352006 plasmid digested with XbaI
S K1352006 plasmid digested with SpeI
P K1352006 plasmid digested with PstI
EP K1352006 plasmid digested with EcoRI and PstI
XS K1352006 plasmid digested with XbaI and Spe1


DNA Sequencing

The recombinant plasmid was Sanger-sequenced with the following sequencing primers. “G101” is a reverse primer, the rest are forward primers.

“G101” (attaccgcctttgagtgagc)
“G100” (tgccacctgacgtctaagaa)
“35 INP-SEQ 1” (ccgattcattaatgcagctgg)
“36 INP-SEQ 2” (gaggttgctgttgccgac)
“37 INP-SEQ 3” (ggtgtggaagccgacattc)

The plasmid insert was found to be exactly as desired. Highlighted below is the MCS excerpt from the “G101” primer results (“G101” is a reverse primer which is why the sequence is in reverse complement).



Conclusions

The part is RFC 10 compatible. The construct sequence was produced exactly as designed; the full FLAG-tag – with BglII and HindIII sites – is present at the N-terminus of the linker sequence after INP.