Genetics

Genetics

<b>4. Genetics</b> <b>4.1. Genetic Tools</b> A variety of genetic tools are available for methanotrophs and have been successfully applied for decades.  Replicating vectors are broad host-range vectors, most commonly of the IncP type, but other replicons have also been successful. The tools include vectors for general cloning, expression, promoter screening, chromosomal insertion, and gene deletion (see Table 4.1) <b>Table 4.1. Vectors Used in Methanotrophs</b> <table width="100%" border="0" cellspacing="0" cellpadding="0"> <tbody> <tr> <td valign="bottom" width="16%"><b>Vector</b></td> <td valign="bottom" width="17%"><b>Inc group</b></td> <td valign="bottom" width="10%"><b>Selective markers</b></td> <td valign="bottom" width="27%"><b>Strains</b></td> <td valign="bottom" width="15%"><b>References</b></td> <td valign="bottom" width="12%"><b>Availability</b></td> </tr> <tr> <td valign="bottom" width="16%"><b>Conjugation helper vectors</b></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"></td> <td valign="bottom" width="15%"></td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pRK2013</td> <td valign="bottom" width="17%">ColEI</td> <td valign="bottom" width="10%">Km</td> <td valign="bottom" width="27%"><i>Methylosinus sp</i>. 6<i></i></td> <td valign="bottom" width="15%">(<a title="Toukdarian, 1984 #257" href="/wiki/references#_ENREF_182">Toukdarian and Lidstrom, 1984</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" nowrap="nowrap" width="16%"></td> <td valign="bottom" nowrap="nowrap" width="17%"></td> <td valign="bottom" nowrap="nowrap" width="10%"></td> <td valign="bottom" width="27%"><i>Methylomonas </i>16a</td> <td valign="bottom" width="15%">(<a title="Sharpe, 2007 #226" href="/wiki/references#_ENREF_162">Sharpe et al., 2007</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" nowrap="nowrap" width="16%">pRK2073</td> <td valign="bottom" nowrap="nowrap" width="17%"></td> <td valign="bottom" nowrap="nowrap" width="10%">Sm</td> <td valign="bottom" width="27%"><i>Methylomicrobium alcaliphilum </i>20Z<i></i></td> <td valign="bottom" width="15%">(<a title="Mustakhimov, 2010 #184" href="/wiki/references#_ENREF_134">Mustakhimov et al., 2010</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">strain S17-1</td> <td valign="bottom" width="17%">(tra genes in chromosome)</td> <td valign="bottom" nowrap="nowrap" width="10%"></td> <td valign="bottom" width="27%"><i>Methylosinus trichosporium </i>OB3b<i></i></td> <td valign="bottom" width="15%">(<a title="Martin, 1995 #165" href="/wiki/references#_ENREF_129">Martin and Murrell, 1995</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="15%">(<a title="Stolyar, 1999 #246" href="/wiki/references#_ENREF_177">Stolyar et al., 1999</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"><i>Methylosinus trichosporium </i>OB3b<i>, Methylomicrobium album </i>BG8<i> (Methylobacter albus </i>BG8<i>), Methylocystis parvus </i>OBBP<i></i></td> <td valign="bottom" width="15%">(<a title="Lloyd, 1999 #160" href="/wiki/references#_ENREF_126">Lloyd et al., 1999a</a>; <a title="Lloyd, 1999 #161" href="/wiki/references#_ENREF_127">Lloyd et al., 1999b</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"><i>Methylomicrobium alcaliphilum </i>20Z<i></i></td> <td valign="bottom" width="15%">(<a title="Mustakhimov, 2010 #184" href="/wiki/references#_ENREF_134">Mustakhimov et al., 2010</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"></td> <td valign="bottom" nowrap="nowrap" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"><i>Methylocella sylvestris </i>SC2<i></i></td> <td valign="bottom" width="15%">(<a title="Theisen, 2005 #254" href="/wiki/references#_ENREF_180">Theisen et al., 2005</a>)</td> <td valign="bottom" nowrap="nowrap" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"><b>Replicating vectors</b></td> <td valign="bottom" nowrap="nowrap" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" nowrap="nowrap" width="27%"></td> <td valign="bottom" nowrap="nowrap" width="15%"></td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pVK100</td> <td valign="bottom" width="17%">Inc P</td> <td valign="bottom" width="10%">Tc, Km</td> <td valign="bottom" width="27%"><i>Methylosinus trichosporium </i>OB3b<i>, Methylomicrobium album </i>BG8<i> (Methylobacter albus </i>BG8<i>), Methylocystis parvus </i>OBBP<i></i></td> <td valign="bottom" width="15%">(<a title="Lloyd, 1999 #160" href="/wiki/references#_ENREF_126">Lloyd et al., 1999a</a>; <a title="Lloyd, 1999 #161" href="/wiki/references#_ENREF_127">Lloyd et al., 1999b</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"><i>Methylosinus sporium</i></td> <td valign="bottom" width="15%">(<a title="Ali, 2006 #2" href="/wiki/references#_ENREF_11">Ali et al., 2006</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pHM1</td> <td valign="bottom" width="17%">Inc Q</td> <td valign="bottom" width="10%">Sm, Km</td> <td valign="bottom" width="27%"><i>Methylosinus trichosporium </i>OB3b<i></i></td> <td valign="bottom" width="15%">(<a title="Lloyd, 1999 #160" href="/wiki/references#_ENREF_126">Lloyd et al., 1999a</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pSRK-Km</td> <td valign="bottom" width="17%">pBBR1 deriv</td> <td valign="bottom" width="10%">Km</td> <td valign="bottom" width="27%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="15%">(<a title="Welander, 2012 #275" href="/wiki/references#_ENREF_197">Welander and Summons, 2012</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"><b>Expression vectors</b></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"></td> <td valign="bottom" width="15%"></td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pGD500</td> <td valign="bottom" width="17%">IncP; reporter lacZ</td> <td valign="bottom" nowrap="nowrap" width="10%">Tc</td> <td valign="bottom" width="27%"><i>Methylomicrobium album </i>BG8<i> (Methylobacter albus </i>BG8<i>)</i></td> <td valign="bottom" width="15%">(<a title="Chistoserdova, 1994 #43" href="/wiki/references#_ENREF_45">Chistoserdova et al., 1994</a>)</td> <td valign="bottom" width="12%">not available</td> </tr> <tr> <td valign="bottom" width="16%">pMHA203</td> <td valign="bottom" width="17%">IncP; reporter GFP; sigma54 promoter</td> <td valign="bottom" nowrap="nowrap" width="10%">Km</td> <td valign="bottom" width="27%"><i>Methylocella silvestris </i>BL2<i></i></td> <td valign="bottom" width="15%">(<a title="Theisen, 2005 #254" href="/wiki/references#_ENREF_180">Theisen et al., 2005</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pBHR1</td> <td valign="bottom" width="17%">IncP; pCAT promoter</td> <td valign="bottom" nowrap="nowrap" width="10%">Km</td> <td valign="bottom" width="27%"><i>Methylomonas </i>16a<i></i></td> <td valign="bottom" width="15%">(<a title="Sharpe, 2007 #226" href="/wiki/references#_ENREF_162">Sharpe et al., 2007</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"><b>Promoter cloning vectors</b></td> <td valign="bottom" width="17%"></td> <td valign="bottom" nowrap="nowrap" width="10%"></td> <td valign="bottom" width="27%"></td> <td valign="bottom" width="15%"></td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pPROBE-NT derivative</td> <td valign="bottom" width="17%">pBBR1; reporter GFP</td> <td valign="bottom" nowrap="nowrap" width="10%">Km</td> <td valign="bottom" width="27%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="15%">(<a title="Csaki, 2003 #58" href="/wiki/references#_ENREF_53">Csaki et al., 2003</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pMHA200</td> <td valign="bottom" width="17%">IncP, ColE1; reporter GFP</td> <td valign="bottom" width="10%">Km</td> <td valign="bottom" width="27%"><i>Methylocella silvestris </i>BL2<i></i></td> <td valign="bottom" width="15%">(<a title="Theisen, 2005 #254" href="/wiki/references#_ENREF_180">Theisen et al., 2005</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pMHA010</td> <td valign="bottom" width="17%">ColE1 (integrative);mob;  reporter GFP</td> <td valign="bottom" width="10%">Gm</td> <td valign="bottom" width="27%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="15%">(<a title="Ali, 2009 #3" href="/wiki/references#_ENREF_10">Ali and Murrell, 2009</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pMHA012</td> <td valign="bottom" width="17%">ColE1 (integrative);mob;  reporter GFP</td> <td valign="bottom" width="10%">Km</td> <td valign="bottom" width="27%"></td> <td valign="bottom" width="15%">(<a title="Ali, 2009 #3" href="/wiki/references#_ENREF_10">Ali and Murrell, 2009</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pMHA020</td> <td valign="bottom" width="17%">ColE1 (integrative); reporter; reporter XylE</td> <td valign="bottom" width="10%">Gm</td> <td valign="bottom" width="27%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="15%">(<a title="Ali, 2009 #3" href="/wiki/references#_ENREF_10">Ali and Murrell, 2009</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pMHA030</td> <td valign="bottom" width="17%">ColE1 (integrative); mob;  reporter; reporter LacZ</td> <td valign="bottom" width="10%">Gm</td> <td valign="bottom" width="27%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="15%">(<a title="Ali, 2009 #3" href="/wiki/references#_ENREF_10">Ali and Murrell, 2009</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pMHA199</td> <td valign="bottom" width="17%">IncP, ColE1; mob; reporter XylE</td> <td valign="bottom" width="10%">Km</td> <td valign="bottom" width="27%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="15%">(<a title="Ali, 2009 #3" href="/wiki/references#_ENREF_10">Ali and Murrell, 2009</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pTSGex</td> <td valign="bottom" width="17%">IncP, ColE1; mob; reporter GFP</td> <td valign="bottom" width="10%">Km</td> <td valign="bottom" width="27%"><i>Methylomicrobium alcaliphilum </i>20Z<i></i></td> <td valign="bottom" width="15%">(<a title="Mustakhimov, 2010 #184" href="/wiki/references#_ENREF_134">Mustakhimov et al., 2010</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"><b>Deletion (suicide) vectors</b></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"></td> <td valign="bottom" width="15%"></td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pBR325</td> <td valign="bottom" width="17%">oriV (ColE1)</td> <td valign="bottom" width="10%">Ap</td> <td valign="bottom" width="27%"><i>Methylosinus sp. </i>6<i></i></td> <td valign="bottom" width="15%">(<a title="Toukdarian, 1984 #257" href="/wiki/references#_ENREF_182">Toukdarian and Lidstrom, 1984</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pBR325mob</td> <td valign="bottom" width="17%">oriV (ColE1)/mob</td> <td valign="bottom" width="10%">Ap</td> <td valign="bottom" width="27%"><i>Methylosinus trichosporium </i>OB3b<i></i></td> <td valign="bottom" width="15%">(<a title="Martin, 1995 #165" href="/wiki/references#_ENREF_129">Martin and Murrell, 1995</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pAYC61</td> <td valign="bottom" width="17%">oriV (ColE1)/mob</td> <td valign="bottom" width="10%">Ap, Tc</td> <td valign="bottom" width="27%"><i>Methylomicrobium album </i>BG8<i> (Methylomonas albus </i>BG8<i>)</i></td> <td valign="bottom" width="15%">(<a title="Berson, 1997 #25" href="/wiki/references#_ENREF_28">Berson and Lidstrom, 1997</a>)</td> <td valign="bottom" width="12%">not available; precursor to pCM184</td> </tr> <tr> <td valign="bottom" width="16%"></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="15%">(<a title="Stolyar, 1999 #246" href="/wiki/references#_ENREF_177">Stolyar et al., 1999</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pK18mob</td> <td valign="bottom" width="17%">oriV (ColE1)/mob</td> <td valign="bottom" width="10%">Km</td> <td valign="bottom" width="27%"><i>Methylosinus trichosporium </i>OB3b<i></i></td> <td valign="bottom" width="15%">(<a title="Stafford, 2003 #236" href="/wiki/references#_ENREF_169">Stafford et al., 2003</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"><i>Methylosinus sporium</i></td> <td valign="bottom" width="15%">(<a title="Ali, 2006 #2" href="/wiki/references#_ENREF_11">Ali et al., 2006</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pBR329mob</td> <td valign="bottom" width="17%">oriV (ColE1)/mob</td> <td valign="bottom" width="10%">Ap, Km</td> <td valign="bottom" width="27%"><i>Methylosinus trichosporium </i>OB3b<i></i></td> <td valign="bottom" width="15%">(<a title="Stafford, 2003 #236" href="/wiki/references#_ENREF_169">Stafford et al., 2003</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pK18mobsacB</td> <td valign="bottom" width="17%">oriV (ColE1)/mob w/sacB</td> <td valign="bottom" width="10%">Km</td> <td valign="bottom" width="27%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="15%">(<a title="Csaki, 2003 #58" href="/wiki/references#_ENREF_53">Csaki et al., 2003</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"><i>Methylosinus trichosporium </i>OB3b<i></i></td> <td valign="bottom" width="15%">(<a title="Semrau, 2013 #224" href="/wiki/references#_ENREF_161">Semrau et al., 2013</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pCM184</td> <td valign="bottom" width="17%">oriV (ColE1)/mob</td> <td valign="bottom" nowrap="nowrap" width="10%">Ap, Tc</td> <td valign="bottom" width="27%"><i>Methylocella sylvestris</i></td> <td valign="bottom" width="15%">(<a title="Chen, 2010 #40" href="/wiki/references#_ENREF_42">Chen et al., 2010</a>)</td> <td valign="bottom" width="12%">Addgene</td> </tr> <tr> <td valign="bottom" width="16%"></td> <td valign="bottom" width="17%"></td> <td valign="bottom" nowrap="nowrap" width="10%"></td> <td valign="bottom" width="27%"><i>Methylomicrobium alcaliphilum </i>20Z<i></i></td> <td valign="bottom" width="15%">(<a title="Mustakhimov, 2010 #184" href="/wiki/references#_ENREF_134">Mustakhimov et al., 2010</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pUC18</td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%">Ap</td> <td valign="bottom" width="27%"><i>Methylocystis </i>SC2<i></i></td> <td valign="bottom" width="15%">(<a title="Baani, 2008 #16" href="/wiki/references#_ENREF_22">Baani and Liesack, 2008</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pJQ200SK</td> <td valign="bottom" width="17%">p15a w/sacB</td> <td valign="bottom" width="10%">Gm</td> <td valign="bottom" width="27%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="15%">(<a title="Welander, 2012 #275" href="/wiki/references#_ENREF_197">Welander and Summons, 2012</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pUTmini-Tn5km</td> <td valign="bottom" width="17%">Mobilizable transposon vector for chromosomal insertion</td> <td valign="bottom" width="10%">Km, Ap</td> <td valign="bottom" width="27%"><i>Methylosinus</i> sp. B4S</td> <td valign="bottom" width="15%">(<a title="Iguchi, 2013 #97B" href="/wiki/references#_ENREF_97B">Iguchi et al., 2013</a>)</td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%"><b>Marker exchange vectors</b></td> <td valign="bottom" width="17%"></td> <td valign="bottom" width="10%"></td> <td valign="bottom" width="27%"></td> <td valign="bottom" width="15%"></td> <td valign="bottom" width="12%"></td> </tr> <tr> <td valign="bottom" width="16%">pGP704-sacB</td> <td valign="bottom" width="17%">IncP (R6K) w/sacB as a selection</td> <td valign="bottom" width="10%">Ap</td> <td valign="bottom" width="27%"><i>Methylomonas </i>16a<i></i></td> <td valign="bottom" width="15%">(<a title="Sharpe, 2007 #226" href="/wiki/references#_ENREF_162">Sharpe et al., 2007</a>)</td> <td valign="bottom" width="12%"></td> </tr> </tbody> </table> &nbsp; <b>4.2 Gene Transfer</b> The most common mode of gene transfer for methanotrophs is conjugation, although electroporation has also been successful in Type II methanotrophs (Table 4.2). The one report of transformation (<a title="Williams, 1971 #280" href="/wiki/references#_ENREF_201">Williams and Bainbridge, 1971</a>) has not been repeated. <b>4.2.1. Conjugation Protocols. </b>Detailed protocols can be obtained in the references noted in Table 4.2. In general, conjugative plasmids are introduced into an <i>E. coli</i> donor strain by transformation.  It is common to use strain S17-1, which has transfer genes integrated into the chromosome.  Alternatively, triparental matings can be used with an <i>E. coli</i> donor strain, the recipient strain, and a helper strain containing a plasmid with transfer genes, such as pRK2013. Strains can be either grown on plates or in liquid cultures, then mixed, normally at a donor:recipient ratio of 1:2 to 1:5 and plated on a mating plate containing diluted rich medium plus the normal methanotroph medium, incubated on a substrate the methanotroph can use, normally methane. Alternatively, the mixture can be filtered and the mating is carried out on a filter placed on a mating plate. Mating time depends on the growth rate of the methanotroph, but 1-2 days is common.  Cells are then transferred to selective plates and allowed to grow out, compared to controls with <i>E. coli</i> not containing the donor plasmid.  Depending on the strain it can take 2-3 weeks for colonies to appear.  If an antibiotic-resistant methanotroph strain is not available, it is common to retain some amount of <i>E. coli</i> on the first transfer to selective plates and in this case, it is necessary to restreak single colonies to confirm they are the methanotroph containing the antibiotic resistance of the plasmid to be transferred.  Many methanotrophs are naturally resistant to 25 µg/ml naladixic acid, and if a nal-sensitive strain of <i>E. coli </i>is used, this can be used as a selective marker. <b>Table 4.2.  Gene Transfer Approaches Used in Methanotrophs</b> <table width="553" border="0" cellspacing="0" cellpadding="0"> <tbody> <tr> <td valign="bottom" width="123"><b>Technique</b></td> <td valign="bottom" width="238"><b>Strains</b></td> <td valign="bottom" width="192"><b>References</b></td> </tr> <tr> <td valign="bottom" width="123">Transformation</td> <td valign="bottom" width="238"><i>Methylococcus capsulatus</i></td> <td valign="bottom" width="192">(<a title="Williams, 1971 #280" href="/wiki/references#_ENREF_201">Williams and Bainbridge, 1971</a>)</td> </tr> <tr> <td valign="bottom" width="123">Conjugation</td> <td valign="bottom" width="238"><i>Methylosinus sp. 6</i></td> <td valign="bottom" width="192">(<a title="Toukdarian, 1984 #257" href="/wiki/references#_ENREF_182">Toukdarian and Lidstrom, 1984</a>)</td> </tr> <tr> <td valign="bottom" width="123"></td> <td valign="bottom" width="238"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="192">(<a title="Stolyar, 1999 #246" href="/wiki/references#_ENREF_177">Stolyar et al., 1999</a>)</td> </tr> <tr> <td valign="bottom" width="123"></td> <td valign="bottom" width="238"><i>Methylosinus trichosporium </i>OB3b<i></i></td> <td valign="bottom" width="192">(<a title="Lloyd, 1999 #160" href="/wiki/references#_ENREF_126">Lloyd et al., 1999a</a>)</td> </tr> <tr> <td valign="bottom" width="123"></td> <td valign="bottom" width="238"><i>Methylomicrobium album </i>BG8<i> (Methylomonas albus </i>BG8<i>)</i></td> <td valign="bottom" width="192">(<a title="Lloyd, 1999 #161" href="/wiki/references#_ENREF_127">Lloyd et al., 1999b</a>)</td> </tr> <tr> <td valign="bottom" width="123"></td> <td valign="bottom" width="238"><i>Methylocystis parvus </i>OBBP<i></i></td> <td valign="bottom" width="192">(<a title="Lloyd, 1999 #161" href="/wiki/references#_ENREF_127">Lloyd et al., 1999b</a>)</td> </tr> <tr> <td valign="bottom" width="123"></td> <td valign="bottom" width="238"><i>Methylocella sylvestris </i>BL2<i></i></td> <td valign="bottom" width="192">(<a title="Theisen, 2005 #254" href="/wiki/references#_ENREF_180">Theisen et al., 2005</a>)</td> </tr> <tr> <td valign="bottom" nowrap="nowrap" width="123"></td> <td valign="bottom" width="238"><i>Methylomonas </i>16a<i></i></td> <td valign="bottom" width="192">(<a title="Sharpe, 2007 #226" href="/wiki/references#_ENREF_162">Sharpe et al., 2007</a>)</td> </tr> <tr> <td valign="bottom" nowrap="nowrap" width="123"></td> <td valign="bottom" width="238"><i>Methylomicrobium alcaliphilum </i>20Z<i></i></td> <td valign="bottom" width="192">(<a title="Mustakhimov, 2010 #184" href="/wiki/references#_ENREF_134">Mustakhimov et al., 2010</a>)</td> </tr> <tr> <td valign="bottom" width="123">Electroporation</td> <td valign="bottom" width="238"><i>Methylocystis </i>SC2<i></i></td> <td valign="bottom" width="192">(<a title="Baani, 2008 #16" href="/wiki/references#_ENREF_22">Baani and Liesack, 2008</a>)</td> </tr> <tr> <td valign="bottom" width="123"></td> <td valign="bottom" width="238"><i>Methylocella silvestris </i>BL2</td> <td valign="bottom" width="192">(<a title="Crombie, 2011 #57" href="/wiki/references#_ENREF_52">Crombie and Murrell, 2011</a>)</td> </tr> <tr> <td valign="bottom" width="123">Transposon mutagenesis</td> <td valign="bottom" width="238"><i>Methylosinus sp. </i>6<i></i></td> <td valign="bottom" width="192">(<a title="Toukdarian, 1984 #257" href="/wiki/references#_ENREF_182">Toukdarian and Lidstrom, 1984</a>)</td> </tr> <tr> <td valign="bottom" width="123"></td> <td valign="bottom" width="238"><i>Methylomonas </i>16a<i></i></td> <td valign="bottom" width="192">(<a title="Sharpe, 2007 #226" href="/wiki/references#_ENREF_162">Sharpe et al., 2007</a>)</td> </tr> </tbody> </table> &nbsp; <b>4.3.  Expression of Methanotroph Genes in Alternate Hosts</b> A number of methanotroph genes have been successful expressed in alternate hosts (Table 4.3).  However, the pMMO subunits are very toxic to <i>E. coli</i> and have not yet been successfully expressed, with the exception of engineered fragments of the soluble domain of the <i>pmoB</i> subunit (<a title="Balasubramanian, 2010 #18" href="/wiki/references#_ENREF_23">Balasubramanian et al., 2010</a>). It is often difficult to clone these subunits in <i>E. coli</i>, and low copy, low expression vectors are sometimes needed. For the sMMO, neither methane oxidation nor propylene oxidation activity have been demonstrated for sMMO expressed in alternate hosts (<a title="Jahng, 1996 #309" href="/wiki/references#_ENREF_99">Jahng et al., 1996</a>; <a title="West, 1992 #277" href="/wiki/references#_ENREF_198">West et al., 1992</a>), and it has been shown in <i>E. coli</i> that the hydroxylase component is inactive (<a title="West, 1992 #277" href="/wiki/references#_ENREF_198">West et al., 1992</a>) However, active sMMO has been expressed in methanotroph hosts not containing sMMO (<a title="Lloyd, 1999 #160" href="/wiki/references#_ENREF_126">Lloyd et al., 1999a</a>). <b>Table 4.3.  Expression of Methanotroph Genes in Alternate Hosts</b> <table width="100%" border="0" cellspacing="0" cellpadding="0"> <tbody> <tr> <td valign="bottom" width="16%"><b>Alternate host</b></td> <td valign="bottom" width="36%"><b>Strain from which genes originated</b></td> <td valign="bottom" width="22%"><b>Genes expressed</b></td> <td valign="bottom" width="24%"><b>References</b></td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli</i></td> <td valign="bottom" width="36%"><i>Methylococcus capsulatus </i>Bath,<i> Methylomicrobium album </i>BG8<i> (Methylomonas albus </i>BG8)<i></i></td> <td valign="bottom" width="22%"><i>mxaF</i> (<i>moxF</i>) from T7</td> <td valign="bottom" width="24%">(<a title="Stephens, 1988 #244" href="/wiki/references#_ENREF_175">Stephens et al., 1988</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli</i></td> <td valign="bottom" width="36%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="22%"><i>glnA</i> in pBR322</td> <td valign="bottom" width="24%">(<a title="Cardy, 1990 #37" href="/wiki/references#_ENREF_39">Cardy and Murrell, 1990</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli </i></td> <td valign="bottom" width="36%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="22%"><i>mmoB, mmoC</i> from T7</td> <td valign="bottom" width="24%">(<a title="West, 1992 #277" href="/wiki/references#_ENREF_198">West et al., 1992</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli </i></td> <td valign="bottom" width="36%"><i>Methylomonas sp. </i>A4<i></i></td> <td valign="bottom" width="22%"><i>mxaF</i> (<i>moxF</i>) from T7</td> <td valign="bottom" width="24%">(<a title="Waechterbrulla, 1993 #270" href="/wiki/references#_ENREF_192">Waechterbrulla et al., 1993</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli</i></td> <td valign="bottom" width="36%"><i>Methylomicrobium album </i>BG8<i> (Methylomonas albus </i>BG8)<i></i></td> <td valign="bottom" width="22%"><i>mxaACKLD</i> from T7</td> <td valign="bottom" width="24%">(<a title="Arps, 1995 #15" href="/wiki/references#_ENREF_19">Arps et al., 1995</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli</i></td> <td valign="bottom" width="36%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="22%"><i>mopE</i> from T7</td> <td valign="bottom" width="24%">(<a title="Fjellbirkeland, 2001 #95" href="/wiki/references#_ENREF_78">Fjellbirkeland et al., 2001</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli </i></td> <td valign="bottom" width="36%"><i>Methylomicrobium alcaliphilum </i>20Z<i></i></td> <td valign="bottom" width="22%"><i>ectA</i> (ectoine biosyn) from T7</td> <td valign="bottom" width="24%">(<a title="Reshetnikov, 2005 #207" href="/wiki/references#_ENREF_150">Reshetnikov et al., 2005</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli </i></td> <td valign="bottom" width="36%"><i>Methylomicrobium alcaliphilum </i>20Z<i></i></td> <td valign="bottom" width="22%"><i>ectABC</i> (ectoine biosyn) from T7</td> <td valign="bottom" width="24%">(<a title="Reshetnikov, 2006 #206" href="/wiki/references#_ENREF_149">Reshetnikov et al., 2006</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli</i></td> <td valign="bottom" width="36%"><i>Methylomicrobium alcaliphilum </i>20Z<i></i></td> <td valign="bottom" width="22%"><i>pfp</i> (phosphofructokinase) from T7</td> <td valign="bottom" width="24%">(<a title="Reshetnikov, 2008 #208" href="/wiki/references#_ENREF_151">Reshetnikov et al., 2008</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli</i></td> <td valign="bottom" width="36%"><i>Methylomicrobium alcaliphilum </i>20Z<i></i></td> <td valign="bottom" width="22%"><i>ectR</i> (regulator) from T7</td> <td valign="bottom" width="24%">(<a title="Mustakhimov, 2010 #184" href="/wiki/references#_ENREF_134">Mustakhimov et al., 2010</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli</i></td> <td valign="bottom" width="36%"><i>Methylococcus capsulatus </i>Bath<i></i></td> <td valign="bottom" width="22%"><i>fba</i> from T7</td> <td valign="bottom" width="24%">(<a title="Rozova, 2010 #215" href="/wiki/references#_ENREF_155">Rozova et al., 2010</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli</i></td> <td valign="bottom" width="36%"><i>Methylosinus trichosporium </i>OB3b<i></i></td> <td valign="bottom" width="22%"><i>fba</i> from T7</td> <td valign="bottom" width="24%">(<a title="Rozova, 2012 #217" href="/wiki/references#_ENREF_156">Rozova et al., 2012</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>E. coli</i></td> <td valign="bottom" width="36%"><i>Methylomicrobium alcaliphilum </i>20Z<i></i></td> <td valign="bottom" width="22%">ATP-dep fructokinase</td> <td valign="bottom" width="24%">(<a title="But, 2012 #34" href="/wiki/references#_ENREF_37">But et al., 2012</a>)</td> </tr> <tr> <td valign="bottom" width="16%"><i>M. parvus </i>OBBP<i></i></td> <td valign="bottom" width="36%"><i>Methylococcus capsulatus</i> Bath <i>Methylosinus trichosporium</i> OB3b</td> <td valign="bottom" width="22%">sMMO</td> <td valign="bottom" width="24%">(<a title="Lloyd, 1999 #160" href="/wiki/references#_ENREF_126">Lloyd et al., 1999a</a>)</td> </tr> <tr> <td valign="bottom" nowrap="nowrap" width="16%"><i>M. album </i>BG8</td> <td valign="bottom" width="36%"><i>Methylococcus capsulatus</i> Bath <i>Methylosinus trichosporium</i> OB3b</td> <td valign="bottom" width="22%">sMMO</td> <td valign="bottom" nowrap="nowrap" width="24%">(<a title="Lloyd, 1999 #160" href="/wiki/references#_ENREF_126">Lloyd et al., 1999a</a>)</td> </tr> </tbody> </table>