Difference between revisions of "Team:London Biohackspace/experiments/miraculin"

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                   <h2>Experiments</h2>
 
                   <h2>Experiments</h2>
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                       <h3>SLiCE <i>Ex vivo</i> DNA assembly</h3>
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                       <h3>EXPRESSING MIRACULIN IN <i>S. CEREVISIAE</i></h3>
  
 
                         <h4>Introduction</h4>
 
                         <h4>Introduction</h4>
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                             <p>We developed a DNA assembly system purely based on parts homology, which only uses <i>E. coli </i> lysate to carry out the reaction. Our work builds on the previous research on lysate based assembly methods, in the particular <a href="http://nar.oxfordjournals.org/content/40/8/e55.long">SLiCE</a> and <a href="http://journal.frontiersin.org/article/10.3389/fbioe.2013.00012/abstract"><i>Ex vivo</i></a>. The concept is very similar to that of a Gibson method: the parts to be assembled contain an overlapping homology region, which allows homologous recombination to occur. While the Gibson assembly utilises an expensive piece of kit, containing a 3' to 5' exonuclease, a DNA polymerase to fill the gaps and a ligase to seal the nick. The <i>Ex vivo</i>, as we like to call it "E.G., or <i>E. coli </i> gratiae" only uses <i>E. coli lysate </i> to carry out this reaction. The lysate in fact does contain all the cellular machinery necessary to recognise a homology and to repair DNA. This process if facilitated when the lysate contains three lambda proteins, which can be easily expressed in the strains used to produce it. In addition to normal lysate, this system was tested using a lysate of cells expressing lamda proteins. These are the same protein that allow Lambda Red Recombineering Knock-Outs, <i> i.e. </i> Gam, Exo and Beta, which respectively protect linear DNA from RecBCD nuclease activity, cleave DNA 3' to 5' and promote annealing of complementary single strands.
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                             <p>The BioBrick encoding the (S. cerevisiae codon-optimised) Miraculin protein coding sequence will be synthesized and ligated into a pSB1C3 plasmid prior to submission to the iGEM registry. Once this part has been synthesized, we aim to use the SureVector expression vector assembly kit to create a plasmid capable of expressing the Miraculin protein. In order to achieve this we will need to PCR amplify the coding sequence from the pSB1C3 plasmid to create a sequence containing the required SureVector overlap sequences. This DNA fragment can then be used as our gene of interest during the SureVector plasmid assembly process. The assembled expression vector will also contain a yeast autonomous replication sequence (yARS) as well as a LEU2 auxotrophic marker to allow for expression in Leucine deficient strains of S. cerevisiae. Expression of the gene will be regulated through the use of the S. cerevisiae X-Gal Galactose inducible promoter provided with the SureVector kit. The SureVector expression plasmid also contains a His-tagging sequence that we can use to characterise Miraclin expression once a yeast strain has be transformed with the plasmid.</p>
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                            <br><br>
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                            We decided to test the efficacy of SLiCE for the assembly of parts only based on a short flanking homology. This homology is roughly equivalent to that of the biobrick prefix and suffix. This means that a two part assembly, of the insert - such as a gblock - into the standard pSB1C3 vector would only require one Seamless Ligation step. This avoids the standard (and costly!) digestion/ligation steps that usually are required for the biobrick assembly.<br>
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                            We tested this approach by ligating the standard J04450 RFP generator to pSB1C3 using 22bp and 21bp of homology (biobrick prefix and suffix :) ) and successfully achieved pink, ligated colonies.
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                            </p>
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                        <br><br>
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                         <h4>Materials and methods</h4>
 
                         <h4>Materials and methods</h4>
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                            <p> <br>
 
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                            <b>SLiCE & SPLiCE reactions we used</b>
 
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                            Standard SLiCE reaction: <br>
 
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                            - 50–200 ng linear vector<br>
 
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                            - Appropriate amount of insert DNA in a 1 : 1 to 10 : 1 molar ratio of insert to vector,<br>
 
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                            - 1 ul 10X SLiCE buffer (500mMTris–HCl (pH 7.5 at 25?C), 100mM MgCl2, 10mM ATP, 10mM DTT)<br><br>
 
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                            Quick Reaction with Quick T4 Ligase Buffer (50ul reaction): <br>
 
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                            - 50–200 ng linear vector<br>
 
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                            - Appropriate amount of insert DNA in a 3:1 molar ratio of insert to vector
 
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                            - 25ul of 2X T4 ligase buffer (66mM Tris-HCL. 10mM MgCl2, 1mM Dithiothreitol, 1mM ATP, 7.5% Polyethylene glycol (PEG6000), pH 7.6 @ 25°C) <br>
 
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                            Quick SPLiCE (10ul or 50ul reaction):
 
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                            - 50-200ng linear vector <br>
 
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                            - Appropriate amount of insert DNA in a 3:1 molar ratio of insert to vector <br>
 
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                            - 1ul of <i>E. coli</i> lysate (see below for preparation) <br>
 
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                            - 1ul of T4 ligase buffer (1x)
 
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                            - 1ul PEG 8000 (3% final concentration)
 
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<br><br>
 
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                            <b> Preparing the lysate (lambda protein express)</b> <br>
 
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                            - Transform cells with plasmid pKD46 (or use strain with genomic copy <i> e.g.</i> EcNR2)<br>
 
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                            - Pick colony and grow O/N colture<br>
 
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                            - Dilute 1:100 in 50ml (for 5ml of lysate, 1ml is 1000 reactions)<br>
 
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                            - Grow to OD 1.0 (tips for DIY OD measurement <a href="https://wiki.london.hackspace.org.uk/view/Project:OD600_Measurement"><i>here</i></a> )<br>
 
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                            - Spin 50ml for 10 minutes at 400g and resuspend in 5ml of Tris-NaCl 10mM, transfer in eppendorf tubes<br>
 
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                            - Spin the 1ml aliquots 8000g for 1 min and remove supernatant<br>
 
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                            - [We then flash froze the pellets and continued the reaction a month later, we suggest not to wait a month and skip to the next step]<br>
 
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                            - Resuspend the cells in 100ul of lysis buffer for 10 minutes: we used B-Per by thermo fisher - any equivalent will do.
 
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                            - Spin 15000g for 5 minutes<br>
 
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                            - Carefully removed the supernatant and mix with 100ul of sterile glycerol to a final volume of 50% glycerol<br>
 
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                            - It should maintain efficiency after months in -20 according to the original papers<br>
 
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                            - We call it Ex-vivoase/Smashcoli/Splicease, your call :)<br>
 
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<br><br>
 
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                            Note that this is the protocol we used for the first test and it was done in a UCL lab, this is easily optimisable and we are working on making it more hackspace friendly.
 
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                            <b>  Amplifying the parts </b>
 
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                            </p>
 
 
                         <h4>Results</h4>
 
                         <h4>Results</h4>
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                            <p>
 
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                          Initial reaction, SPLiCE
 
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                            </p>
 
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                         <h4> Discussion</h4>     
 
                         <h4> Discussion</h4>     
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                        <p> Reaction conditions,
 
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                        improved "quick ex vivo",
 
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                        prefix suffix homology and homology length.
 
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                        control to exclude background
 
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                        </p>
 
 
                        
 
                        
  

Revision as of 23:19, 18 September 2015

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  • Lab Book

Experiments

EXPRESSING MIRACULIN IN S. CEREVISIAE

Introduction

The BioBrick encoding the (S. cerevisiae codon-optimised) Miraculin protein coding sequence will be synthesized and ligated into a pSB1C3 plasmid prior to submission to the iGEM registry. Once this part has been synthesized, we aim to use the SureVector expression vector assembly kit to create a plasmid capable of expressing the Miraculin protein. In order to achieve this we will need to PCR amplify the coding sequence from the pSB1C3 plasmid to create a sequence containing the required SureVector overlap sequences. This DNA fragment can then be used as our gene of interest during the SureVector plasmid assembly process. The assembled expression vector will also contain a yeast autonomous replication sequence (yARS) as well as a LEU2 auxotrophic marker to allow for expression in Leucine deficient strains of S. cerevisiae. Expression of the gene will be regulated through the use of the S. cerevisiae X-Gal Galactose inducible promoter provided with the SureVector kit. The SureVector expression plasmid also contains a His-tagging sequence that we can use to characterise Miraclin expression once a yeast strain has be transformed with the plasmid.

Materials and methods

Results

Discussion

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