Team:Reading/Project

From 2014.igem.org

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<tr><td><h3><font color="#558e2b"> Project Description </font></h3></td>
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<tr><td><h3><font color="#558e2b">A note on protocols</font></h3></td>
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<td> <h3 id="poverview"><font color="#558e2b"> Contents </font></h3></td>
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<td> <h3><font color="#558e2b">Contents</font></h3></td>
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<p>Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project.</p>
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Biological photovoltaics (<a href="https://en.wikipedia.org/wiki/Biological_photovoltaics">BPVs</a>) have the potential to provide a more sustainable alternative to traditional photovoltaics. However, current implementations have not been able to provide sufficient electron output to be viable alternatives to solar panels. Our project aims to increase output by redirecting electron flow in our chosen cyanobacteria, <i>Synechocystis</i> sp. PCC 6803 (henceforth <i>Synechocystis</i>).
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<!-- Contents -->
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<li><a href="#poverview">Project Overview</a></li>
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<li><a href="#">A Note on Protocols</a>
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<li><a href="#problem">The Problem</a></li>
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<li><a href="#protocols">Protocols</a>
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<li><a href="#syne">Why <i>Synechocystis</i>?</a></li>
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<ul>
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<li><a href="#aims">Project Aims</a></li>
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    <li><a href="#prot1">Miniprep</a>
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<li><a href="#methods">Methods</a></li>
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    <li><a href="#prot2">Glycerol stock</a>
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    <li><a href="#prot3">Optical Density</a>
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    <li><a href="#prot4">PCR</a></li>
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    <li><a href="#prot5">Transformation (E. coli)</a>
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    <li><a href="#prot6">Nanodrop</a>
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    <li><a href="#prot7">BG-11 plates</a>
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    <li><a href="#prot8">Cyanobacteria Transformation</a>
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    <li><a href="#prot9">Biobrick Assembly</a>
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</ul>
<li><a href="#references">References</a></li>
<li><a href="#references">References</a></li>
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<li><a href="#acknowledge">Acknowledgements</a></li>
</ol>
</ol>
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<td colspan="3"><h3 id="problem"> <font color="#558e2b">The Problem</font> </h3>
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<td colspan="3"><h3 id=”prot1”><font color="#558e2b">Protocols</font> </h3>
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Solar photovoltaics are one of the main sources of renewable energy and are likely to become more widely used in the future . However, they are far from perfect. Although cyanobacteria have been predicted to have photosynthesis efficiency of around 11%<sup>1</sup> (maximum recorded current produced is 11pA/cell<sup>2</sup>), compared to 10-32%<sup>3</sup> for traditional photovoltaics, BPVs could have several benefits. As they are made from living organisms, BPVs are capable of self-repair. Furthermore, they consume carbon dioxide during photosynthesis and are cheaper to produce, so have been proposed for use in lower income countries where less energy is required per person<sup>2</sup>. Though BPVs are worth pursing as an alternative to traditional photovoltaics, their potential electrical output is yet to be realised. Our project seeks to improve upon current levels and so make BPVs more viable.
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<!--Miniprep -->
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<p><font color="#558e2b"><i>Isolation of plasmid DNA from bacteria (miniprep)</i></font></p>
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<ol>
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  <li>Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes
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  <li>Discard supernatant by pipetting off ensuring not to disturb the pellet
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  <li>Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes
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  <li>Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times
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  <li>Centrifuge at 13,000 rpm for 5 minutes
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  <li>Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate
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  <li>Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm
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  <li>Discard the flow through
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  <li>Add 500 µl of wash solution to the column
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  <li>Centrifuge for 1 minute at 13,000 rpm
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  <li>Discard flow through
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  <li id=”prot2”>Add 500 µl of wash solution to the column
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  <li>Centrifuge for 1 minute at 13,000 rpm
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  <li>Discard flow through
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  <li>Centrifuge for 1 minute at 13,000 rpm
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  <li>Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube
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  <li>Add 35 µl of ultrapure water. Do not touch the membrane with the pipette
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  <li>Incubate at room temperature for 2 minutes
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  <li>Centrifuge for 2 minutes at 13,000 rpm
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</ol>
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<!-- Glycerol stock -->
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<td colspan="3"><h3 id="syne"><font color="#558e2b">Why <i>Synechocystis</i>?</font></h3>
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<p><font color="#292929">  
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<p><font color="#558e2b"><i>Making glycerol stock</i></font></p>
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Photolithoautotrophic microorganisms derive their energy from sunlight, their electrons from inorganic sources, and their carbon from inorganic carbon sources. This is particularly useful as sunlight, water and carbon dioxide can provide these three vital resources respectively. Algae and cyanobacteria are both candidates for use in BPVs, but the latter provide several advantages: they are easier to manipulate genetically, the grow more quickly and have an increasing number of tools available<sup>4</sup>. <i>Synechocystis</i> was the first cyanobacteria to have its genome completely sequenced<sup>5</sup>, has been used extensively in mathematical modelling of cyanobacteria<sup>4</sup>, and seems to be the most commonly used in species BPVs.
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<p>This protocol is adapted from 2 freely available protocols<sup><a href="#references">1</a>, <a href="#references">2</a></sup>. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet</p>
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</font></p>
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<ol>
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</td>
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  <li>Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips
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  <li>Centrifuge at 13,000 rpm for 2 minutes
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  <li>Discard supernatant
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  <li>Add 0.5 ml of 60% glycerol stock
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  <ol style="list-style: lower-roman outside">
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    <li>240 ml of glycerol
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    <li>160 ml nano pure water
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    <li>mix together and autoclave
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  </ol>
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  <li>freeze at -80℃
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</ol>
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<!-- Optical Density -->
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<br>
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<p><font color="#558e2b"><i>Taking optical density of culture</i></font></p>
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<p>Recommendation for taking OD for monitoring growth is either OD<sub>730</sub><sup><a href="#references">3</a></sup> or OD<sub>750</sub><sup><a href="#references">4</a></sup>. Some recommend taking OD<sub>730</sub> at no higher than 0.4 because of problems with light scattering<sup><a href=”#references”>4</a></sup>. We chose to measure at growth OD<sub>750</sub> to keep in line with other high-profile papers on Synechocystis<sup><a href="#references">4</a></sup>.</p>
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<ol>
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<li>Set spectrophotometer to measure at OD<sub>750</sub>
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<li>Blank with 1 ml of BG-11 in a cuvette
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<li>Measure 1 ml of culture
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<li>If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11
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</ol>
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<p>Converting OD<sub>750</sub> to cell density For conversion of cell densities to numbers of cells, we have used the relationship OD750 = 1 (a.u) corresponding to 1.6 x 10<sup>8</sup> cells mL<sup><a href="#references">5</a></sup>.</p>
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<!-- PCR -->
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<td colspan="3"><h3 id="aims"><font color="#558e2b">Project Aims</font></h3>
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<br>
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<p><font color="#292929">  
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<p><font color="#558e2b"><i>PCR</i></font></p>
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To improve output in <i>Synechocystis</i> we have targeted key points in the photosynthetic electron transport chain (PETC) to increase the number electrons available, as this has been used successfully in the past<sup>6</sup>, and are attempting to improve the transfer of these electrons to the anode. The electrically insulating outer membrane (OM) appears to be the major limiter in preventing higher electron output, so methods to improve the release of electrons are very important. In general, electrons in BPVs may be donated to the anode through direct contact with the OM, or at a distance by using a soluble mediator or through other methods such as bacterial nanowires<sup>7</sup>. Although it is unclear exactly which of these mechanisms are used by <i>Synechocystis</i><sup>2</sup>, it seems direct contact is the most likely<sup>8</sup>. As direct contact, mediators and nanowires could all potentially be used, our project will target all 3 types of transfer.
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<p>PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.</p>
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</font></p>
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<p> Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage. </p>
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<br />
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<p>2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix <sup><a href="#references">6</a></sup></p>
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<p><font color="#292929">  
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<p>PCR program program as follows:</p>
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The diagram below shows a simplified view of the photosynthetic and respiratory electron transport chains in <i>Synechocystis</i>. The cytoplasmic membrane at the top only contains the respiratory electron transport chain (RETC), while the thylakoid membrane below contains both the respiratory and photosynthetic transport chains. A significant feature of photosynthesis in cyanobacteria is that these two transport chains share several components. Furthermore, soluble mediators (shown in turquoise) can diffuse through the membrane to shuffle electrons between proteins, so the two chains are highly interlinked. The aspects of the PETC that we are targeting are shown in red; for more detail on these, see the <a href="#methods">methods</a> section.
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<ol>
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</font></p>
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<li> Start:
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</td>
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<ul>
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</tr>
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<il>95℃ for 30 seconds
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<tr>
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</ul>
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<td colspan="3">
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<li>35 cycles:
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  <img align=centre src="https://static.igem.org/mediawiki/2014/a/ab/Photosystem_coloured.png" width="900px">
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<ul>
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</td>
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95℃ for 10 seconds
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</tr>
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56℃ for 15 seconds
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72℃ for 70 seconds
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</ul>
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<li>Final
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<ul>
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72℃ for 5 minutes
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68℃ for 10 minutes
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</ul>
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</ol>
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<p>All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol<sup><a href="#references">7</a></sup></p>
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<p><font color="#558e2b"><i>Transformation into E. coli</i></font></p>
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<p>
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<ol>
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<li>Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf
 +
<li>Add 5 ul of DNA using a sterile pipette tip
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<li>Flick the tubes to mix and then store on ice for 30 minutes
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<li>Heat shock in a water bath at 42℃ for 1 minute
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<li>Incubate on ice for 5 minutes
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<li>Add 450 ul of SOC (we used LB instead)
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<li>Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm
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<li>Invert Eppendorfs containing the cells several times
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<li>Plate out and incubate overnight at 37℃
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</ol>
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<p> Notes on transformation efficency</p>
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<p>Expected transformation efficiency is 1 x 10<sup>6</sup> cfu/ug of pUC19 DNA, but we should expect a 2-fold decrease in efficiency due to use of LB instead of SOC (a derivative of super optimal broth, SOB). We should also expect a decrease because we thawed the frozen competent cells at a temperature above 0ºC. Ideally they should be thawed on ice, or by hand if needed <sup><a href="#references">8</a></sup>.</p>
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<!-- Nanodrop -->
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<td colspan="3"><h3 id="methods"><font color="#558e2b">Methods</font></h3>
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<br>
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<p><font color="#558e2b"><i>Nanodrop</i></font></p>
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<p><font color="#558e2b"><i>Targeting the PETC</i></font></p>
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<p>Nanodrop is used to check concentration in DNA often from a miniprep</p>
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<p><font color="#292929">
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<ol>
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Increasing the number of electrons available in the plastoquinone pool is one way of increasing electron output in <i>Synechocystis</i>. This can be done by removing the exit points of the PETC, namely the terminal oxidases and FNR. The former has been shown to increase electron output in the dark phase of diurnal growth cycle by removing the 3 major terminal oxidases<sup>6</sup>, while knocking out FNR seems to prove fatal to the cell as FNR is crucial for cell growth and survival. The removal of FNR ceases the movement of electrons from photosystem I to NADP<sup>+</sup> that is vital for the energy transfer in the cell.  The Flv proteins are terminal oxidases used by <i>Synechocystis</i> and other cyanobacteria that remove electron by reducing oxygen without producing reactive oxygen species (ROS), unlike other terminal oxidases<sup>9</sup>. They are therefore very important in controlling the redox state of the photosystems. 4 Flv proteins are present in <i>Synechocystis</i>; these form Flv1:3 and Flv2:4 heterodimers that attach to PSII and PSI respectively. They appear to provide an electron sink during sunlight<sup>10</sup>, with Flv2:4 providing photoprotection<sup>11</sup> and Flv1:3 allowing survival under fluctuating light<sup>12</sup>. We will create knockouts of both dimers to try and increase the number of electrons available for release; this should increase electron output in light phase of growth cycle, possibly complementing the triple major terminal oxidase mutant.
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<li>Set the Nanodrop to measure DNA
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</font></p>
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<li>Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank
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<li>Add 2 ul of sample and measure. Measurement should be in ng/ul
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</ol>
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<br />
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<!-- BG-11 plates -->
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<br>
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<p><font color="#558e2b"><i>BG-11 plates</i></font></p>
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<p>BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.</p>
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<ol>
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<li>Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise
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<li>If making kanamycin plates cool to ~55℃ and add 50 ug/ml
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<li>Agar melted in steamer and kept at 55℃ until needed for pouring
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</ol>
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<p> If making a kanamycin cap:</p>
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<ol>
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<li>0.6% agar w/v
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<li>Cool BG-11 to ~55ºC and add 0.5mg/ml kan
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<li>Add ~3ml to a plain BG-11 plate with transformed colonies on it
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<li>Leave for >1 week for Kan selection to occur
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</ol>
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<p><font color="#558e2b"><i>Increasing pili</i></font></p>
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<!-- Cyanobacteria Transformation -->
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<p><font color="#292929">  
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<br>
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Bacterial nanowires are modified pili that are capable of transferring electrons out of the cell. They have only become known about fairly recently, with the first discovery in <i>Geobacter sulfereducans</i> in 2005<sup>13</sup>. Since then they appear to have been identified in <i>Shewanella oneidensis</i> strain MR-1 and <i>Synechocystis sp</i> PCC 6803<sup>14</sup>, the strain of cyanobacteria we are using.
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<p><font color="#558e2b"><i>Cyanobacteria Transformation</i></font></p>
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</font></p>
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<p>Protocol to transform DNA into cyanobacteria</p>
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<p><font color="#292929">  
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<ol>
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Since 2006 there has been a considerable amount of work on nanowires, but mainly focusing on <i>Geobacter spp.</i> and <i>S. oneidensis</i>. To our knowledge, no further published work has confirmed or elaborated on the possibility of nanowires in <i>Synechocystis</i>. As such, despite the large role they play in some other species and that the method of electron transfer out of <i>Synechocystis</i> is essentially unknown<sup>2</sup>, we cannot reasonably target nanowires as the core of our project. However, increasing the production of large pili, one of two types that can be seen on our strain<sup>15</sup>, could increase aggregation in biofilms (a benefit in itself as it could increase the number of bacteria growing directly on the anode), but it is possible that they will also increase the number of nanowires if they exist. Furthermore, a similar alteration that affects the number of pili has caused <i>Synechocystis</i> to no longer be transformable due to entanglement of the pili<sup>16</sup>, a possible added biosafety benefit. Conversely, hyperpiliation could cause our bacteria to become more transformable, a serious biosafety issue we will be taking into consideration.
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<li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days
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</font></p>
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<li>Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant
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<p><font color="#292929">  
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<li>Repeat step 2
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To cause hyperpiliation, we will be inserting another copy of the PilA1 gene (one of the main pili components) along with a strong upstream promoter, Ptrc<sup>10</sup>, to initiate high-level expression<sup>17</sup>, and deleting the PilT1 gene, an ATPase that is involved in pilus retraction<sup>18</sup>.
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<li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant
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</font></p>
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<li>Add 200ml fresh BG-11
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<br />
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<li>4ug plasmid A DNA is added (at a concentration of at least 100ng/ul)
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<p><font color="#558e2b"><i>Producing endogenous mediators</i></font></p>
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<li>Incubate for 24 under light on a shaker at ~100 rpm
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<p><font color="#292929">  
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<li>Plate the full 200ml and leave for 1-2 days
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Mediators are redox molecules that can accept and donate electrons. They can therefore carry charge and, if soluble, can transport electrons from the cell to the anode. Mediators have been added to many BPVs to increase output, but this is no longer common practice<sup>2</sup>. Mediators are often toxic so can potentially damage the cell; it is also thought to be less sustainable if mediator has to constantly be added to the BPV.
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<li>Add 3-4ml kan 0.6% agar BG-11
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</font></p>
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<li>Leave under light for >1 week
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<p><font color="#292929">
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</ol>
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Endogenous mediators are those produced by the cell itself. They therefore do not need to be added, making it a more sustainable solution. Flavins are an example of an endogenous mediator. Previous iGEM teams have produced soluble mediators for expression in cyanobacteria; we hope to improve upon these parts.
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</font></p>
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<!-- Biobrick Assembly -->
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<br />
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<br>
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<p><font color="#558e2b"><i>Biosafety</i></font></p>
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<p><font color="#558e2b"><i>Biobrick Assembly</i></font></p>
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<p><font color="#292929">
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<p>Protocol for inserting a construct into a plasmid backbone. In the case of BioBrick this will commonly be pSB1C3. Biobrick has 2 stages, a digestion stage and a ligation stage:</p>
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Safety considerations have been embedded in synthetic biology since the beginnings of the field. As such there are several important, detailed reviews of biosafety<sup>19–21</sup>, but almost all of these focus on <i>Escherichia coli</i>. They also focus on the use of plasmids that are maintained extra-chromosomally, rather than chromosomal modifications. Although <i>E. coli</i> and <i>Synechocystis</i> are both gram negatives, they pose significantly different risks, and very different technologies are used for modifying them. We will address the major differences and draw attention to the safety implications that follow from these.
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<ol>
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</font></p>
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<li>In a PCR tube add 20 ul of water and either 5 ul of your construct or 2 ul of plasmid backbone
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<p><font color="#292929">
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<li>To the same tube add 2.5 ul of NED buffer
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During our project we will be considering the consequences of each of our modifications, and the potential safety problems and solutions faced in applying our technology. We will assume that the technology will make it to market, so will be used in areas where they could feature, such as on the roofs of houses.  
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<li>Add appropriate restriction enzymes. In our case 1 ul of EcoR1 and 1 ul of Pst1
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</font></p>
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<li>Incubate the tubes at 37℃ for 15 minutes
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<li>Then incubate at 80℃ for 20 minutes
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<li>Add 5 ul of water to a new tube followed by 2 ul of the digested construct and backbone
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<li>Add 2 ul of 10X T4 DNA ligase restriction buffer
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<li>Add 1 ul of T4 DNA ligase
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<li>Incubate at room temperature for 10 minutes
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<li>Incubate reaction mixture at 80℃ for 20 minutes. This step inactivates the enzyme
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</ol>
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<p>This DNA can now be used for transformation</p>
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<!-- References Section -->
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<tr>
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<td colspan="3"><h3><font color="#558e2b">References</font></h3>
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<p>1. http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</p>
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<p>2. http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</p>
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<p>3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At <http://link.springer.com/protocol/10.1007/978-1-60761-925-3_22>. Accessed 20/08/2014.</p>
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<p>4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).</p>
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<p>5 - Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p>
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<p>6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p>
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<p>http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf</p>
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<p>https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987</p>
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<p>Everyone we need to thank for help with protocols.</p>
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<ol>
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<li>Brenner, M. P. Engineering Microorganisms for Energy Production. (U.S. Department of Energy, 2006).</li>
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<li>Bradley, R. W., Bombelli, P., Rowden, S. J. L. & Howe, C. J. Biological photovoltaics: intra- and extra-cellular electron transport by cyanobacteria. Biochem. Soc. Trans. 40, 1302–1307 (2012).</li>
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<li>Crabtree, G. W. & Lewis, N. S. Solar energy conversion. Phys. Today 60, 37–42 (2007).</li>
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<li>Berla, B. M. et al. Synthetic biology of cyanobacteria: unique challenges and opportunities. Front. Microbiol. 4, (2013).</li>
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<li>Kaneko, T. et al. Sequence Analysis of the Genome of the Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803. II. Sequence Determination of the Entire Genome and Assignment of Potential Protein-coding Regions. DNA Res. 3, 109–136 (1996).</li>
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<li>Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).</li>
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<li>Malvankar, N. S. & Lovley, D. R. Microbial Nanowires: A New Paradigm for Biological Electron Transfer and Bioelectronics. ChemSusChem 5, 1039–1046 (2012).</li>
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<li>Cereda, A. et al. A Bioelectrochemical Approach to Characterize Extracellular Electron Transfer by Synechocystis sp. PCC6803. PLoS ONE 9, (2014).</li>
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<li>Vermaas, W. F. Photosynthesis and Respiration in Cyanobacteria. (2001). at <http://onlinelibrary.wiley.com/doi/10.1038/npg.els.0001670/abstract></li>
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<li>Allahverdiyeva, Y. et al. Interplay between Flavodiiron Proteins and Photorespiration in Synechocystis sp. PCC 6803. J. Biol. Chem. 286, 24007–24014 (2011).</li>
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<li>Zhang, P., Allahverdiyeva, Y., Eisenhut, M. & Aro, E.-M. Flavodiiron Proteins in Oxygenic Photosynthetic Organisms: Photoprotection of Photosystem II by Flv2 and Flv4 in Synechocystis sp. PCC 6803. PLoS ONE 4, e5331 (2009).</li>
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<li>Allahverdiyeva, Y. et al. Flavodiiron proteins Flv1 and Flv3 enable cyanobacterial growth and photosynthesis under fluctuating light. Proc. Natl. Acad. Sci. 110, 4111–4116 (2013).</li>
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<li>Reguera, G. et al. Extracellular electron transfer via microbial nanowires. Nature 435, 1098–1101 (2005).</li>
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<li>Gorby, Y. A. et al. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc. Natl. Acad. Sci. 103, 11358–11363 (2006).</li>
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<li>Yoshihara, S. & Ikeuchi, M. Phototactic motility in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Photochem. Photobiol. Sci. 3, 512–518 (2004).</li>
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<li>Nakasugi, K., Svenson, C. J. & Neilan, B. A. The competence gene, comF, from Synechocystis sp. strain PCC 6803 is involved in natural transformation, phototactic motility and piliation. Microbiology 152, 3623–3631 (2006).</li>
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<li>Huang, H.-H., Camsund, D., Lindblad, P. & Heidorn, T. Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res. gkq164 (2010). doi:10.1093/nar/gkq164</li>
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<li>Okamoto, S. & Ohmori, M. The Cyanobacterial PilT Protein Responsible for Cell Motility and Transformation Hydrolyzes ATP. Plant Cell Physiol. 43, 1127–1136 (2002).</li>
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<li>Wright, O., Stan, G.-B. & Ellis, T. Building-in biosafety for synthetic biology. Microbiology 159, 1221–1235 (2013).</li>
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<li>Schmidt, M. & de Lorenzo, V. Synthetic constructs in/for the environment: Managing the interplay between natural and engineered Biology. FEBS Lett. 586, 2199–2206 (2012).</li>
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<li>Moe-Behrens, G. H. G., Davis, R. & Haynes, K. A. Preparing synthetic biology for the world. Microbiotechnology Ecotoxicol. Bioremediation 4, 5 (2013).</li>
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Revision as of 22:50, 15 October 2014

University of Reading
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A note on protocols

Contents

Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project.


  1. A Note on Protocols
  2. Protocols
  3. References
  4. Acknowledgements

Protocols


Isolation of plasmid DNA from bacteria (miniprep)

  1. Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes
  2. Discard supernatant by pipetting off ensuring not to disturb the pellet
  3. Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes
  4. Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times
  5. Centrifuge at 13,000 rpm for 5 minutes
  6. Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate
  7. Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm
  8. Discard the flow through
  9. Add 500 µl of wash solution to the column
  10. Centrifuge for 1 minute at 13,000 rpm
  11. Discard flow through
  12. Add 500 µl of wash solution to the column
  13. Centrifuge for 1 minute at 13,000 rpm
  14. Discard flow through
  15. Centrifuge for 1 minute at 13,000 rpm
  16. Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube
  17. Add 35 µl of ultrapure water. Do not touch the membrane with the pipette
  18. Incubate at room temperature for 2 minutes
  19. Centrifuge for 2 minutes at 13,000 rpm

Making glycerol stock

This protocol is adapted from 2 freely available protocols1, 2. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet

  1. Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips
  2. Centrifuge at 13,000 rpm for 2 minutes
  3. Discard supernatant
  4. Add 0.5 ml of 60% glycerol stock
    1. 240 ml of glycerol
    2. 160 ml nano pure water
    3. mix together and autoclave
  5. freeze at -80℃

Taking optical density of culture

Recommendation for taking OD for monitoring growth is either OD7303 or OD7504. Some recommend taking OD730 at no higher than 0.4 because of problems with light scattering4. We chose to measure at growth OD750 to keep in line with other high-profile papers on Synechocystis4.

  1. Set spectrophotometer to measure at OD750
  2. Blank with 1 ml of BG-11 in a cuvette
  3. Measure 1 ml of culture
  4. If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11

Converting OD750 to cell density For conversion of cell densities to numbers of cells, we have used the relationship OD750 = 1 (a.u) corresponding to 1.6 x 108 cells mL5.


PCR

PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.

Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage.

2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix 6

PCR program program as follows:

  1. Start:
      95℃ for 30 seconds
  2. 35 cycles:
      95℃ for 10 seconds 56℃ for 15 seconds 72℃ for 70 seconds
  3. Final
      72℃ for 5 minutes 68℃ for 10 minutes

All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol7


Transformation into E. coli

  1. Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf
  2. Add 5 ul of DNA using a sterile pipette tip
  3. Flick the tubes to mix and then store on ice for 30 minutes
  4. Heat shock in a water bath at 42℃ for 1 minute
  5. Incubate on ice for 5 minutes
  6. Add 450 ul of SOC (we used LB instead)
  7. Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm
  8. Invert Eppendorfs containing the cells several times
  9. Plate out and incubate overnight at 37℃

Notes on transformation efficency

Expected transformation efficiency is 1 x 106 cfu/ug of pUC19 DNA, but we should expect a 2-fold decrease in efficiency due to use of LB instead of SOC (a derivative of super optimal broth, SOB). We should also expect a decrease because we thawed the frozen competent cells at a temperature above 0ºC. Ideally they should be thawed on ice, or by hand if needed 8.


Nanodrop

Nanodrop is used to check concentration in DNA often from a miniprep

  1. Set the Nanodrop to measure DNA
  2. Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank
  3. Add 2 ul of sample and measure. Measurement should be in ng/ul

BG-11 plates

BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.

  1. Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise
  2. If making kanamycin plates cool to ~55℃ and add 50 ug/ml
  3. Agar melted in steamer and kept at 55℃ until needed for pouring

If making a kanamycin cap:

  1. 0.6% agar w/v
  2. Cool BG-11 to ~55ºC and add 0.5mg/ml kan
  3. Add ~3ml to a plain BG-11 plate with transformed colonies on it
  4. Leave for >1 week for Kan selection to occur

Cyanobacteria Transformation

Protocol to transform DNA into cyanobacteria

  1. Make a fresh culture to OD730=0.2 to 0.3 and grow for 3 days
  2. Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant
  3. Repeat step 2
  4. Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant
  5. Add 200ml fresh BG-11
  6. 4ug plasmid A DNA is added (at a concentration of at least 100ng/ul)
  7. Incubate for 24 under light on a shaker at ~100 rpm
  8. Plate the full 200ml and leave for 1-2 days
  9. Add 3-4ml kan 0.6% agar BG-11
  10. Leave under light for >1 week

Biobrick Assembly

Protocol for inserting a construct into a plasmid backbone. In the case of BioBrick this will commonly be pSB1C3. Biobrick has 2 stages, a digestion stage and a ligation stage:

  1. In a PCR tube add 20 ul of water and either 5 ul of your construct or 2 ul of plasmid backbone
  2. To the same tube add 2.5 ul of NED buffer
  3. Add appropriate restriction enzymes. In our case 1 ul of EcoR1 and 1 ul of Pst1
  4. Incubate the tubes at 37℃ for 15 minutes
  5. Then incubate at 80℃ for 20 minutes
  6. Add 5 ul of water to a new tube followed by 2 ul of the digested construct and backbone
  7. Add 2 ul of 10X T4 DNA ligase restriction buffer
  8. Add 1 ul of T4 DNA ligase
  9. Incubate at room temperature for 10 minutes
  10. Incubate reaction mixture at 80℃ for 20 minutes. This step inactivates the enzyme

This DNA can now be used for transformation

References

1. http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf

2. http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria

3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At . Accessed 20/08/2014.

4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).

5 - Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.

6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.

http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf

https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987

Acknowledgements

Everyone we need to thank for help with protocols.

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