TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was used to express a nourseothricin (<em>nou</em>/<em>nat</em>) resistance gene and a gene encoding a silaffin precursor TpSil3p-APEX2 fusion protein in <em>Thalassiosira pseudonana </em>(<em>Tp</em>) strain <a href="https://ncma.bigelow.org/ccmp1335?___SID=U#.WAQoEvkrL4Y" target="_blank">CCMP1335</a> using conjugation by largely following Karas et al. (2015). In addition to the protocol, I am listing some of my observations working with this diatom species. Please check the 'Before start' and 'Guidelines' sections for more information including a list of <em>Tp</em> episomes I've constructed and which are available upon request. For additional background on my overacrching experimental aim, please refer to <a href="http://openwetware.org/images/c/c1/Turnsek_EMBL_16%27.pdf" target="_blank">this document</a>. </p>
<p> </p>
<p>1. E. V. Armbrust, The Genome of the Diatom Thalassiosira Pseudonana: Ecology, Evolution, and Metabolism. <em>Science</em>. <strong>306</strong>, 79–86 (2004).</p>
<p>2. B. J. Karas <em>et al.</em>, Designer diatom episomes delivered by bacterial conjugation. <em>Nat. Commun.</em> <strong>6</strong>, 6925 (2015).</p>
<p> </p>
<p>Keeping in mind that diatom episomes contain a yeast centromere, it would be very interesting to see if conjugation could be adapted to organisms beyond diatoms.</p>

[BEFORE_START]
<p><span style="text-decoration: underline;"><strong>[1] Preparation of donor bacteria</strong></span></p>
<p> </p>
<p>1.1. Any <em>E. coli </em>strain will probably work just fine, but I stuck with the one from the original publication: <a href="http://www.epibio.com/applications/genomic-library-construction-kits-gene-pcr-cloning-kits/competent-cells/transformax-epi300-electrocompetent-e-coli" target="_blank">EPI300</a> (Epicentre, catalog # EC300110).</p>
<p>1.2. Electroporate pTA-Mob into EPI300 cells and select on LB agar plates w/ 10 or 20 µg/mL gentamycin.</p>
<p> </p>
<p><strong>NOTE:</strong></p>
<p>Please refer to the following publication to learn more about the mobilization plasmid pTA-Mob: T. A. Strand, R. Lale, K. F. Degnes, M. Lando, S. Valla, A new and improved host-independent plasmid system for RK2-based conjugal transfer. <em>PLoS One</em>. <strong>9</strong>, e90372 (2014).</p>
<p> </p>
<p>1.3. Make pTA-Mob-containing strain chemically competent.</p>
<p>1.4. Tranform your engineered pTpPuc3 cargo plasmid into pTA-Mob-containing cells.</p>
<p> </p>
<p><strong>NOTE:</strong></p>
<p>If you already have a working <em>Tp </em>plasmid that you successfully transformed via biolistics or some other method, you can easily convert it into a conjugation-compatible vector. Refer to Karas et al. (2015) for details on how to perform this conversion. </p>
<p> </p>
<p>1.5. Select double transformants on LB agar plates with 10 or 20 µg/mL gentamycin and 50 or 100 µg/mL kanamycin.</p>
<p>1.6. Store plates at 4<sup>o</sup>C.</p>
<p> </p>
<p> </p>
<p><span style="text-decoration: underline;"><strong>[2] Preparation of L1 medium and L1 1% agar plates</strong></span></p>
<p> </p>
<p>I was using <a href="https://ncma.bigelow.org/mkl150l#.WAQ4__krL4Y" target="_blank">L1 Medium Kit</a> and <a href="https://ncma.bigelow.org/seabh10l?___SID=U" target="_blank">Gulf of Maine Seawater</a>, both from NCMA, to make my liquid medium and plates.</p>
<p> </p>
<p><strong>NOTE:</strong></p>
<p>I also had success with <a href="https://ncma.bigelow.org/media/pdf/NCMA%20algal%20medium%20ESAW.pdf" target="_blank">ESAW</a>, <a href="https://ncma.bigelow.org/media/wysiwyg/Algal_recipes/NCMA_algal_medium_f_2_1.pdf" target="_blank">f/2</a> and <a href="http://www.nbrc.nite.go.jp/NBRC2/NBRCMediumDetailServlet?NO=963" target="_blank">Daigo's IMK</a> media, but never tested them for the full conjugation protocol. Daigo's IMK can be ordered on <a href="http://www.e-reagent.com/uh/Myp.do?now=1423869442383" target="_blank">this website</a> by using a serch term "IMK" (select catolog # 392-01331).</p>
<p> </p>
<p><span style="text-decoration: underline;"><em>2.1 L1 medium</em></span></p>
<p>I would usually make batches of 1L by combining seawater, N, P and Si sources from the kit followed by autoclaving, cooling down to ~50<sup><span style="font-size: 10.8333px;">o</span></sup>C and adding vitamins and antibiotics. The resulting medium had pH ~8.5. Before starting a diatom culture I would adjust the pH with HCl to ~8.0. Starting pH anywhere between 7.8 and 8.2 should be fine. All my liquid cultures were grown in a batch mode at constant light, ~18<sup><span style="font-size: 10.8333px;">o</span></sup>C and moderate shaking (~100 rpm). I never buffered the medium and/or aerate with air or CO<sub>2</sub>.</p>
<p> </p>
<p><strong>NOTE:</strong> </p>
<p>The resulting medium contain some precipitates due to Si and seawater-derived compunds. You can use a combination of microwaving and filter sterilization instead which should prevent precipitation.</p>
<p>  </p>
<p><span style="text-decoration: underline;"><em>2.2 1/2xL1 1% agar plates w/ 5% LB</em></span></p>
<p>I would always prepare 0.5L and make ~12 plates 40 mL each. For 0.5L combine and mix seawater, N, P and Si sources from the kit, add agar and 5% LB. Autoclave, cool down to ~50 <sup>0</sup>C and add vitamins. </p>
<p> </p>
<p><strong>NOTES:</strong> </p>
<p>- 5% LB is <strong><span style="text-decoration: underline;">v/v not w/v</span></strong>. I had success preparing plates w/v, but <span style="text-decoration: underline;"><strong>v/v is what you should use</strong></span>. In other words: 1.25 g LB powder per 1 L.  </p>
<p>- 1/2xL1: cut all N, P, Si and vitamin volumes from the kit in half.    </p>
<p>- I had success with Bacto Agar and <a href="http://www.sigmaaldrich.com/catalog/product/sial/05038?lang=en&amp;region=US" target="_blank">Sigma's high purity agar</a>.</p>
<p> </p>
<p><span style="text-decoration: underline;"><em>2.3.1/2xL1 1% agar plates w/ 100 µg/mL nourseothricin</em></span></p>
<p>Same as above except the LB part; add nourseothricin when cooled down to ~50 <sup>0</sup>C. My nourseothricin stocks are 200 mg/mL.   </p>
<p> </p>
<p><strong>NOTE:</strong></p>
<p>I observed a lot of background - false positive - colonies on plates with only 50 µg/mL nourseothricin so I switched to 100 µg/mL. You can read more about nourseothricin on the <a href="https://www.jenabioscience.com/images/625337ef95/NTC_superior_selection_tool.pdf" target="_blank">following website</a> which I found very useful.</p>
<p> </p>

[GUIDELINES]
<p><span style="text-decoration: underline;"><strong>[1] 'Crashing phenomenon' in <em>Tp</em><em> </em>cultures</strong></span></p>
<p> </p>
<p>In my experience <em>Tp</em><em> </em>CCMP1335 has to be subcultured frequently - every ~2 weeks - to avoid 'crashing'. I usually dilute my cultures 1:10 in fresh L1 medium. By 'crashing' I mean sudden take over of what I believe is a naturally associated bacterium in the culture. This usually happens in the stationary phase over the course of 1 or 2 days during which brown diatom culture becomes milky due to bacteria. It could be a standard contamination issue, but I've pretty consistently observed this phenomenon even after ordering completely fresh stocks from the NCMA. I analyzed the bacterium via 16S sequencing and some simple growth experiments and confirmed it was a <em>Pseudomonas </em>species. You can read more about how I've done this <a href="http://openwetware.org/images/d/dc/JT_Project_Ljubljana_SHORT.pdf" target="_blank">here</a> and find the paper associated with my 16S and <em>rpoD </em>(<em>Pseudomonas-</em>specific) analysis <a href="http://www.microbiologyresearch.org/docserver/fulltext/ijsem/65/2/424_ijs065201.pdf?expires=1476901544&amp;id=id&amp;accname=guest&amp;checksum=4F8E64933EBF327662C898160A970486" target="_blank">here</a>.  </p>
<p> </p>
<p>Interestingly, such <em>Tp</em>-<em>Pseudomonas </em>interplay was <a href="http://aem.asm.org/content/35/4/791.full.pdf" target="_blank">already observed in the late 1970's</a>.     </p>
<p> </p>
<p> </p>
<p><span style="text-decoration: underline;"><strong>[2] Episomes for <em>Tp</em> conjugation available upon request </strong></span></p>
<p> </p>
<p>All episomes used in my conjugation experiments including their maps are available upon request. Send an email to turnsek@fas.harvard.edu, turnsek@ucsd.edu or jturnsek@jcvi.org and we'll go from there/I can provide more information. Here is a list of available episomes:</p>
<p> </p>
<p>pTpPuc3 - empty nourseothricin resistance cassette-containing episome for <em>T. pseudonana</em> conjugation</p>
<p>eGFP - 2 different enhanced GFP constructs</p>
<p><a href="https://www.dna20.com/products/protein-paintbox#collapse5" target="_blank">DreidelTeal</a> - blue chromogenic protein </p>
<p>TpSil3p-eGFP - frustule-associated silaffin precursor protein TpSil3p tagged with eGFP</p>
<p>TpSil3p-APEX2-FLAG - frustule-associated silaffin precursor protein TpSil3p tagged with APEX2 and FLAG</p>
<p>TpSil3p-APEX2-6xHis - frustule-associated silaffin precursor protein TpSil3p tagged with APEX2 and 6xHis</p>
<p> </p>
<p><strong>NOTE:</strong></p>
<p>All the genes above were cloned into pTpPuc3 Kan<sup>R</sup> backbone via Gibson Assembly starting 36 nucleotides downstream of the HIS3 gene. They are flanked by a constitutive fcp promoter and an associated terminator. Here are their sequences in FASTA format:</p>
<p> </p>
<p>&gt;Tp_fcp_promoter</p>
<p>GCGCTTTTTCCGAGAACTCCCCATAAGTCAACGGCTCCAATCAAGAATGTATCCGACAACGGCGAGCATAGCAACACGTCCGTCTTTGGAGTAGAATCATCATGTTGTGGATGAATACACAGA</p>
<p>TGAATGACATTAAAAGCATGAACATGTTAGAGAGTAGGAGGTAGAGATTGATATGGTAGCATTGCGATGTTTGTTTTTGGTCAGCATATGATGAGTGGATACCAATATGATGAAAGTTGAATCT</p>
<p>CGCGTTTGAGCTCAGCGGTACGTTATTGATCGAAAGTAGCCTGATCAAAATCCTTGGAGAGTACAAGAGGATCAAAGAATCCAGTGGGGGCGATAACTCCAAGCTCGTTCTCAAAGAGGCAA</p>
<p>TGGAGGTAGAAACTCATCCCAGTTGAGAAGAAGTGAAGGCAGTGGCGGTGGCGAAAGCAGAGGCAACGAGGACAGACTTCCTGTGGGTTGATGCAACGAATATTTCCAGAAGGAGAAG</p>
<p>TTTAGAGAGTTGAACCGCTACCTACAATGACAAAGTATCGTATCGATTTTGATGTTGGTTGGTTATGAATTCAAACTGTAAGTTGGATTGTGAGAAGATCAGAAGTTGAACGAACACATCTTTC</p>
<p>CGATCATTCACCTCCACACTGCAACAACACGGTACTTCTTCCGCGGCAGGTCTCTGTCGCCATTCTCTTGTCCTGTTGTTGGCTGTGAGACGAGGAAAGCAACGACAAGTTTCACAAAAGGG</p>
<p>AGTTCCTTTAACGAGATATGTTTTTTATAAAGAGTCCCAATAGAAAGACAAATTGATTCCTCCGTGCAAACGCGCAAATAAACACCACGTCCATTATATCCATATCTTTCAGAGTATCCAACAAGT</p>
<p>GTTGAAGGACAGGTAGTTGAAGTAACGTATCTTCCCCCTCGACTGGATCCATCAACAAGGCGAACAAATCCATTCAACCTCTCATAAATTATCTGATTTACCAAACC </p>
<p> </p>
<p>&gt;Tp_fcp_terminator</p>
<p>ATACTGGATTGGTGAATCAATGAGCCGTAGCACAATGGTTACATTCGGCTAGCTAAGATCCAATGGCAAGGACCAAGTGCTGGAACTTGTTTTGCTTTAGCAGATCTTAGCGTGAGAGGTATT</p>
<p>TGTCCTCTGTCAGGAGTAGATAGTAGATGTTCTTTTTAAACTAAAATGCTAACTGTTCCGAATTCCTCATCGCAGCTAATCCGTACATCAAAAGACAAAATGCTAGGTATGTGTACTACATCTCC</p>
<p>TGTTGCTAGATAAGACATATGATAGGAAACACACCATCAATAGTCATTGTAGCTTTACTTATACTACGCATTTGCACTTTCCCCTGAGTGGCAGAGGCGCATTGAGAAAATCGATCTCAACATAG</p>
<p>TTTATGTAGCATCCCCTAGATCCATTACTTTAAGTCTCCTTCGTCTTTGGTGTAGGCATGTTGGACACAACGAGGTAAAACACAACACAAACAATGTGTCCAGCAAAGTAGTAGCTGCTCCAGT</p>
<p>TCT</p>
<div> </div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.