Difference between revisions of "Team:Goettingen/Results"

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<p><strong>RFP CONSTRUCT</strong></p>
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<p>ESTERASE AND PHOSPHATASE CONSTRUCTS</p>
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<p>RFP (RFP DsRed) was amplified from by PCR (Fig.1). Colonies on a plate were given to us by the Applied and Genomic Microbiology department. Primers contained restriction sites for <em>Kpn</em>I and <em>Sac</em>I in order to make them compatible for insertion into the multiple cloning site of the pBAD A vector.</p>
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<p>Esterase was amplified from pET101_E064 and phosphatase from pTOPOXL_PLP07 by PCR for each enzyme (Fig.1). Both plasmids were given to us by the Applied and Genomic Microbiology department. Primers contained restriction sites <em>Kpn</em>I and <em>Sac</em>I for esterase and <em>Xho</em>I and <em>Pst</em>I for the phosphatase in order to make them compatible for insertion into the multiple cloning site of the pBAD vector .The genes for these two enzymes had been found in screenings of metagenomic libraries.</p>
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<img src=" https://static.igem.org/mediawiki/2015/5/58/PCR_RFP_DsRed_iGEM_Goettingen_2015.jpg" style="; float:right;">
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<p>After purification of the PCR products, they were ligated into pJET by blunt end ligation. This vector serves to clone the enzymes without triggering their activity, which may interact with the vector or the <em>E.coli</em>. In the case of phosphatase, the PCR products needed to be purified by gel extraction, to eliminate left over template plasmids, which could interfere with the transformation.</p>
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<p>After purification of the PCR product it was ligated into pJET1.2 by subcloning (blunt end ligation). This vector serves to clone the fluorescent protein without triggering its activity, which may interact with the expression vector or the chosen <em>E.coli </em>strain.</p>
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<p>Ligation into pJET was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted with a Quiaprep spin Miniprep kit and restricted with either <em>Kpn</em>I and <em>Sac</em>I or <em>Xho</em>I and <em>Pst</em>I, depending on the Enzyme (Fig.2&amp;3).</p>
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<p>Ligation into pJET1.2 was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit and restricted with <em>Kpn</em>I and <em>Sac</em>I (Fig.2).</p>
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<p>Once restrictions showed the correct bands, both pJET containing esterase and phosphatase were sent for sequencing by the G2L laboratory.</p>
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<p>Once restriction controls showed the correct bands, both pJET_RFP_3 and pJET_RFP_7 were sent for sequencing by the G2L laboratory.</p>
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<img src=" https://static.igem.org/mediawiki/2015/1/1d/Restr_pJET_RFP_DsRed_iGEM_Goettingen_2015.jpg" style="; float:right;">
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<p>&nbsp;</p>
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<p><strong>RESULT</strong></p>
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<p>Both sequences were correct. We decided to work with pJET_RFP_3. The plasmid was transformed with <em>E.coli</em> TOP10 and <em>E.coli</em> BL21. Cryocultures were frozen for a strain collection.</p>
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<p>Afterwards the RFP_3 insert was ligated into pBAD A via the T4 ligase system according to the protocol in the methods collection and transformed into <em>E.coli</em> TOP10.</p>
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<p><strong>MICROSCOPY</strong></p>
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<p>To check the red fluorescence transformed <em>E.coli</em> TOP10 were examined by fluorescence microscopy with an excitation at 536 nm. RFP DsRed should show an emission at 582 nm.</p>
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<p>Unfortunately no fluorescence could be detected throughout the whole project. Nevertheless the DNA sequence of the construct is correct. It might be a problem of expression.</p>
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Revision as of 08:10, 16 September 2015



Project Results

Transformation Efficiency Kit, RFP construct (iGEM)

RFP construct

Esterase and Phosphatase construct

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