Difference between revisions of "Team:Berlin diagnostX/Experiments"

Line 279: Line 279:
 
      
 
      
 
      
 
      
 +
<!-- Other Experiments -->
 +
 
<!-- Other Experiments -->
 
<!-- Other Experiments -->
  
Line 287: Line 289:
 
     </div>
 
     </div>
 
</div>
 
</div>
 +
<div class="row">
 +
    <div class="col-12">
 +
        <h3 class="text-center igem_blue mt-4 mb-1">Other Experiments</h3>
 +
        <h5 class="text-center mb-2">Preparation of nucleic acids</h5>
 +
        <p class="text-justify">For our experiments, a large set of different DNAs and RNAs - primers, templates, targets and controls – are required in varied concetrations. Thus, a set of standardized but easily adaptable protocols to extract and purify nucleic acids was established.</p>
 +
    </div>
 
</div>
 
</div>
 +
<div class="row mt-4">
 +
    <div class="col-6">
 +
        <h5 class="text-center igem_blue mb-1">DNA preparation out of E.coli bacteria (Mini-prep, Midi-prep)</h5>
 +
        <p class="text-justify"><strong>Goal:</strong>Frequently used DNA (and RNA coding DNA) is stored as plasmid in apathogenic and nuclease-down-regulating strands of E.coli bacteria and extracted when needed.</p>
 +
        <p class="text-justify"><strong>Description</strong>: Depending of the required quantity, we use different techniques to extract the DNA from the bacteria:</p>
 +
        <ul>
 +
            <li><strong>Miniprep: </strong>Lysis of up to 5mL of bacterial culture and isolation of plasmids via column purification and repeated centrifugation. <br>
 +
            <em>Suitable for desired concentrations between 50-100ng/uL and volumes of up 30uL; mostly used for DNA that is purposed for in vitro transcription</em>.</li>
 +
            <li><strong>Midiprep: </strong>Lysis of up to 250 mL of bacterial culture and isolation of Plasmids via vacuum assisted column purification and high speed centrifugation. <br>
 +
            <em>High throughput method for concentration requirements of 200-400ng/uL and volumes of up to 500-1000uL.</em></li>
 +
        </ul>
 +
    </div>
 +
    <div class="col-6">
 +
        <img class="img-fluid" src="https://static.igem.org/mediawiki/2017/4/4f/T--Berlin_diagnostX--Miniprep%2BVacuum.gif">
 +
    </div>
 +
</div>
 +
<div class="row mt-4">
 +
    <div class="col-6">
 +
        <h5 class="igem_blue text-center pb-1">DNA linearization</h5>
 +
        <p class="text-justify"><strong>Goal:</strong> Both for cell-free expression and in vitro transcription, plasmids need to be transformed to a linearized form.</p>
 +
        <p class="text-justify"><strong>Description</strong> To this end, we use high fidelity restriction enzymes (e.g. EcoRI-HF). The choice of restriction enzyme determines the number of cuts in the plasmid: This can be either one cut to simply linearize the entire plasmid, or two cuts to cut out the plasmid backbone without any insert. The success of the digest can be verified via gel electrophoresis.</p>
 +
    </div>
 +
   
 +
    <div class="col-6">
 +
        <h5 class="igem_blue text-center pb-1">In vitro transcription</h5>
 +
        <p class="text-justify"><strong>Goal:</strong>Obtain specific concentrations of purified target RNA need to be added to the sensor test reaction.</p>
 +
        <p class="text-justify"><strong>Description</strong>The DNA is obtained by transcribing linearized DNA in vitro with a SP6 RNA-Polymerase (RiboMax®, Promega). <br> This kit requires adding linearized template DNA, RNA polymerase, nucleotides and water into a microcentrifuge tube and incubating it at 37 °C. The protocol allows for transcribing RNA in the range of double microgram from smallest concentrations of DNA template in less than one hour. </p>
 +
    </div>   
 +
</div>   
 +
<div class="row">
 +
    <div class="col-12">
 +
        <h5 class="igem_blue text-center pb-1">In vitro transcription</h5>
 +
        <p class="text-justify">We require high RNA concentrations to minimize the volume we have to add to the cell free expression system – too high dilutions significantly decrease its efficiency. <br> <br> To ensure RNA yields with maximal concentration and purity of salts/contaminants, we don’t use column purification systems but rather rely on phenol-chloroform extraction. This method efficiently separates organic and inorganic phases to get rid of any unwanted salt contamination. Moreover the final yield is much higher than with column purification, which mostly reduces final concentration considerably due to column occlusion by contaminants. </p>
 +
    </div>
 +
   
 +
</div>
 +
   
 +
</div>   
 +
 +
 +
<!-- Optimizing Screening -->
 +
 +
<div id="screening_opti">
 +
<div class="row">
 +
    <div class="col-12">
 +
        <h3 class="text-center igem_blue mt-4">Optimizing the Screening Pipeline</h3>
 +
    </div>
 +
</div>
 +
<div class="row mt-4">
 +
    <div class="col-6">
 +
        <p class="text-justify"><strong>Goal:</strong>Establishing the PureExpress® cell free protein expression kits (PureExpress®, NEB) as our main tool for RNA detection and assess its capabilities.</p>
 +
        <p class="text-justify"><strong>Description</strong>The PureExpress®-System is composition of completely recombinant molecules to effectively transcribe any form of DNA into mRNA and then translate this into a protein. To use this system for field diagnostics, it has to meet the following requirements:</p>
 +
        <ul>
 +
            <li>Heat stability up to a temperature of 50°C</li>
 +
            <li>High efficiency even with low volumes of reagents and/or low concentrations</li>
 +
            <li>Stability towards changes in electrolyte concentration/ addition of foreign molecules</li>
 +
            <li>Long-term storability.</li>
 +
        </ul>
 +
    </div>
 +
    <div class="col-6">
 +
        <p class="text-justify">We verified this by evaluating the kinetics of sensor reactions in different experimental conditions, including different temperatures, different reactant concentrations, changes in buffer as well as re-measurements after storage.<br><br>We could show that the system meets all these requirements. Especially noteworthy, we were able to create significant color change in a volume of 2,5 uL which is only 10% of the volume recommended by the manufacturer. Furthermore, we could show that the system is still highly active in concentrations of 40-50%. This makes it possible to use the system with patient samples of only estimated concentration without risking reduced functionality. Last but not least, the system is not vulnerable to possibly interfering molecules, as for example the chlor-phenol-red used  as a reporter dye.
 +
        <br> <br> We concluded that the PureExpress® - System is most suitable for our essay; also due to the fact, that it composed of recombinant parts instead of cell lysate and so has very few variability</p>
 +
    </div>
 +
</div>
 +
<div class="row mt-5">
 +
    <div class="col-12">
 +
        <h5 class="text-center mb-2">Preparation of sensors for iGEM Submission</h5>
 +
    </div>
 +
</div>
 +
<div class="row mt-5">
 +
    <div class="col-12">
 +
        <h5 class="text-left mb-2 igem_blue">Mutagenesis</h5>
 +
        <p class="text-justify"><strong>Goal:</strong>Introduce a silent mutation at position (3181;G>A) in lacZ to remove EcoRI restriction site.</p>
 +
        <p class="text-justify"><strong>Description</strong>We used Q5 mutagenesis kit (NEB) and designed primers using NEBaseChanger software. The software recommended a forward-primer introducing the mutation and a reverse primer with suitable tm to complete the plasmid. Since the software neither provided a ranking of other primer pairs nor detailed information on how the primers are chosen, we designed two more primer pairs introducing the mutation. Mutagenesis was carried out according to the manufacturers protocol with 1ng, 8ng and 20ng of plasmid being used for each primer pair. Successful mutagenesis was confirmed by sequencing.</p>
 +
    </div>
 +
</div>
 +
<div class="row mt-5">
 +
    <div class="col-12">
 +
        <h5 class="text-left mb-2 igem_blue">Cloning into pSB1C</h5>
 +
    </div>
 +
</div>
 +
<div class="row">
 +
    <div class="col-6"><p class="text-justify">All of our experiments were PCR-Based. For this reason, we had to clone our sensors into pSB1C for iGEM-Submission only and were selecting two strategies to reach this goal: </p>
 +
    <ul><li>Cloning of our PCR-Product into pGEM®-T Easy vector and subsequent insertion of the insert to pSB1C via Not1-Restriciton sites.</li><li>Amplifying our PCR-Product using tailed primers introducing the restriction sites SpeI and XbaI. Direct insertion into pSB1C</li></ul>
 +
    <p class="text-justify"><strong>Methods: </strong>For TA-Cloning 5,5 uL of PCR-Product, generated by Phusion PCR according to our SOPs, were incubated with 0,5 uL Units GoTaq® Flexi DNA Polymerase (5U/uL) from Promega, 2ul dATP (2mM), and 2uL GoTaq® Flexi Buffer (5X) for 30 minutes at 70°C. Ligation was performed over night at 4°C using T4-Ligase (M180A Promega) at a molar vector:insert ratio of 1:1 and transformed to ultracompetent DH5a-Cells (NEB). Positive clones were identified by colony PCR using a primer annealing to the T7-promoter as forward-primer and two reverse primers annealing to lacZ. These primers worked well for sequencing the switches already and thus we decided to use them again. <br> <br>FW_T7: GCGAATTAATACGACTCACTATAGGG <br> Rev_Seq1 (310BP-Product): TGAATGGCGAATGGCGCTTTG <br><br>
 +
        Rev_Seq2 (445 BP-Product): CATCTACACCAACGTGACCTATC <br> 5mL bacterial cultures were grown from 4 positive clones per sensor. DNA was isolated the next day using NucleoSpin MiniPrep System (Merchery-Nagel) according to the manufacturer’s instructions. DNA was digested using NotI HF (1U/ug of DNA in CutSmart Buffer) for one hour and heat-inactivated for 20 minutes at 65°C. 2,5 ug of digested DNA was loaded on a gel, bands were excised and cleaned up using ZymoClean™ Gel DNA recovery kit according to the manufacturer’s instructions. Recovery of DNA was low (12-30ng/uL). Never the less we tried to clone the Gel-Purified, dephosphorylated pSB1C-Backbone, which was a kind gift from iGEM Potsdam. Ligation was performed using Quick Ligase (NEB) with a Backbone:Insert Ration of 1:1 and 1:3. Subsequent transformation into ultracompetent DH5a-Cells and growth on Agar plates containing Chloramphenicol did not result in any colonies.       
 +
    </p>
 +
    </div>
 +
    <div class="col-6"><p class="text-justify">For direct cloning into pSB1C using tailed primers, we designed primers introducing XbaI to the 5’ end and SpeI to the 3’ end of the PCR Product. For forward primers two variations were designed to speed experiments up:
 +
<br><br>
 +
FW1: XbaI.T7 GCCGCTTCTAGAtaatacgactcactatagg<br>
 +
FW2: XbaI.T7.6NT GCCGCTTCTAGAGCGAATTAATACGACTCAC<br>
 +
Rev1: SpeI.LacZ.stop  GCCGCTACTAGTTTATTTTTGACACCAGACC<br><br>
 +
 +
 +
Inserts were amplified using Phusion DNA Polymerase (Thermo Fisher) and cleaned up using ZymoClean™ PCR CleanUp-Columns (Zymo Research). Purified PCR products were digested using SpeI and XbaI for 1 hour (all restriction enzymes and Cutsmart Buffer were purchased by NEB).Following heat inactivation at 80°C for 20 minutes the PCR product was purified as described above. The pSB1C-Backbone was cut using the restriction enzymes SpeI and XbaI. To decrease the chance for re-ligation of the vector, we dephosphorylated the vector backbone using calf intestinal alkaline phosphatase (CIP) according to the manufacturer’s instructions (NEB). The digested and phosphorylated backbone plasmid was run on a 1% agarose gel and the aprox. 2.2kb band was cut and purified using the Gel Extraction ZymoClean Kit (Zymo Research). Subsequently, we ligated the purified digested PCR products with the phosphorylated pSB1C backbone using Quick Ligase for 15 minutes at room temperature and transformed the ligated product into ultracompetent DH5a cells. Positive colonies were identified using Colony PCR as mentioned above but with a different forward primer, that binds to the iGEM backbone (VF2 primer ATTACCGCCTTTGAGTGAGC). Bacterial cultures were grown over night and DNA was purified using Quick DNA universal Kit (ZymoResearch) according to the manufacturer’s instructions.</p></div>
 +
</div>
 +
 +
   
 
      
 
      
 +
</div>   
 +
 +
 +
 +
 +
 +
 
</div>
 
</div>
  

Revision as of 22:11, 1 November 2017

Experiments