Wiki/Team:Warsaw/vectors/pKSII+omega-deltaA

From 2008.igem.org

(Difference between revisions)

Revision as of 22:48, 26 October 2008

pKSII + omega-ΔA vector

Description

Author: Michał L., Ewa, Marcin
Date: 9 July 2008
Based on: pKSII+
Resistance: ampicillin
Features: Fusion of omega fragment of β-lactamase and truncated fragment of protein A. Medium-copy vector
Preparation: Protein A coding sequence amplified from pKS-A vector using AP+NotI and AL+link10+homo2 primers. Omega fragment of β-lactamase amplified from pUC19 vector using OmegaL+SacI and OmegaP+link10+homo2. primers. Resulting fragments were then joined together using OmegaL+SacI and AP+NotI primers. This fragment was digested with NotI and SacI and ligated into pKSII+ vector.
As a result we were expecting omega and full length protein A fusion, but in fact we got omega and truncated fragment of protein A (ΔA) fusion.
Usage:

Vector map

Vector sequence

ATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGA
GATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTC
CAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACC
CTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG
CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAA
AGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCAC
CACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCG
CAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG
GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG
TAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCGC
TACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG
GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCG
GTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTA
TCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCG
CTGAGATAGGTGCCTCACTGATTAAGCATTGGGGTGGCGGAGGAAGCGGAGGAGGTGGTT
CTGGAGGTGGAGGTAGCGGGGGTGGGGGTGGTTCGGGTGGAGGTGGTAAAACCGCGGCTC
TTGCGCAACACGATGAAGCCGTGGACAACAAATTCAACAAAGAACAACAAAACGCGTTCT
ATGAGATCTTACATTTACCTAACTTAAACGAAGAACAACGAAACGCCTTCATCCAAAGTT
TAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATG
CTCAGGCGCCGAAAGTAGACGCGAATTCCGCGGGGAAGTCAACCGGCGGCCGCTCTAGAA
CTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCG
AGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCG
TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAAC
ATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACA
TTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCAT
TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCC
TCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA
AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCA
AAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGG
CTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCG
ACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTT
CCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT
TCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGC
TGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT
GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATT
AGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGC
TACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAA
AGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTT
TGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCT
ACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTA
TCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAA
AGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATC
TCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACT
ACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGC
TCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGT
GGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTA
AGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTG
TCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTT
ACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTC
AGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT
ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTC
TGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACC
GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA
CTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAAC
TGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAA
AATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTT
TTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAA
TGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

@@ Line 9: / Line 9: @@
 <b>Date</b>: <a href=https://2008.igem.org/Team:Warsaw/Calendar-Main/9_July_2008>9 July 2008</a><br>
 <b>Based on</b>: <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2B>pKSII+</a><br>
+<b>Resistance</b>: ampicillin<br>
+<b>Features</b>: Fusion of omega fragment of &beta;-lactamase and truncated fragment of protein A. Medium-copy vector<br>
 <b>Preparation</b>: Protein A coding sequence amplified from <A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS-A</a> vector using <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2">AL+link10+homo2</a> primers. Omega fragment of &beta;-lactamase amplified from <a href=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a> vector using <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI">OmegaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaP+link10+homo2">OmegaP+link10+homo2</a>. primers. Resulting fragments were then joined together using <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI">OmegaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> primers. This fragment was digested with NotI and SacI and ligated into <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2B>pKSII+</a> vector. <br>As a result we were expecting omega and full length protein A fusion, but in fact we got omega and truncated fragment of protein A (&Delta;A) fusion.
 <br>