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<!DOCTYPE html>
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<table border="1 " >
<tr>
<th colspan="4 ">Experiment log of 7. 13 & 7. 20, 7. 21 & 7. 23-7. 25 & 7. 26</th>
</tr>
<tr>
<th style="width: 10vw !important;">Date</th>
<th>Member</th>
<th>Experiment</th>
<th>Result</th>
</tr>
<tr>
<td>7. 13</td>
<td>Shi and Tao</td>
<td>Design primer of plasmid linearization and target gene amplification.</td>
<td>Sequence is in the <a href="results#download ">Supplementary Information-1</a>.</td>
</tr>
<tr>
<td>7. 20</td>
<td>Lin , Zheng and Tao</td>
<td>Confect solid YPD medium of 300 mL (detailed prescription is in the <a href="project-PROTOCOL ">protocol</a>).</td>
<td></td>
</tr>
<tr>
<td>7. 21</td>
<td>Lin , Zheng and Tao</td>
<td>Make twenty solid YPD medium.</td>
<td></td>
</tr>
<tr>
<td>7. 23</td>
<td>Tao</td>
<td>Confect solid and liquid LB medium with ampicillin of 300 mL (detailed prescription is in the <a href="project-PROTOCOL ">protocol</a>).<br>
Make 20 medium.<br>
Transformation of DH5α (detailed information is in the <a href="project-PROTOCOL ">protocol</a>. The same below): pFL61 and pFL61-OsNramp5. Breed at constant temperature of 37℃ for 12 h. </td>
<td></td>
</tr>
<tr>
<td>7. 24</td>
<td>Tao</td>
<td>Confect liquid YPD medium of 200 mL. Inoculate <i>△ycf1</i>> on the medium. Bred in shaker at 200 rpm/min, 30 ℃ overnight. <br>
Inoculate DH5α（pFL61 and pFL61-OsNramp5）in the liquid LB amp medium of 140 mL. Bred in shaker at 200 rpm/min, 30 ℃ overnight. </td>
<td></td>
</tr>
<tr>
<td rowspan="2 ">7. 25</td>
<td>Xu, Zhu and Lin
</td>
<td>Confect solid LB medium of 500 mL and liquid LB medium of 1500 mL.
</td>
<td></td>
</tr>
<tr>
<td>Zheng, Liu and Tao</td>
<td>Culture collection: five tubes of DH5α (pFL61) and five tubes of DH5α (pFL61-OsNramp5).<br>
Plasmid Extraction (7. 24) (detailed information is in the <a href="project-PROTOCOL ">protocol</a>, the same below): DH5α (pFL61 and pFL61-OsNramp5).<br> Transformation of DH5α: pFL61 and pFL61-OsNramp5. Bred at constant temperature
of 37 ℃ for 12 h.
</td>
<td></td>
</tr>
<tr>
<td>7. 26</td>
<td>Tao and Liu</td>
<td>Pick transformants in the liquid YPD medium, Bred in shaker at 200 rpm/min, 30℃ for 12 h. <br> Plasmid Extraction: DH5α (pFL61 and pFL61-OsNramp5). <br> PCR: Linearization of pF61 plasmid (model is pF61, primer is L-F/L-R, the same
below).
<br> PCR: Clone of OsNramp5 target section (model is pF61, primer is HR-F/HR-R, the same below. Detailed information is in the <a href="project-PROTOCOL ">protocol</a>)." </td>
<td></td>
</tr>
<tr>
<td></td>
<td>Liu
</td>
<td>"Homologous recombination (linearized pFL61+<i>OsNramp5</i>) ——>pFL61-OsNramp5-HR plasmid. <br> Transformation of DH5α: pFL61-OsNramp5-HR. Bred at constant temperature of 37 ℃ for 12 h. "
</td>
<td></td>
</tr>
</table>
</div>
<br>
<br>
<div class="notebook_cell ">
<table border='1'>
<tr>
<th colspan="4">Experiment log of 7. 27, 7. 28 & 8. 02-8. 06</th>
</tr>
<tr>
<th>Date</th>
<th>Member</th>
<th>Experiment</th>
<th>Result</th>
</tr>
<tr>
<td>7. 27</td>
<td>Tao and Zheng</td>
<td>Confect liquid YPD medium of 200 mL. Inoculate <i>△ycf1</i> on the medium. Bred in shaker at 200 rpm/min, 30 ℃ overnight. PCR: Clone of OsNramp5 target section (detailed information is in the <a href="project-PROTOCOL ">protocol</a>).
Agarose gel (1%) electrophoresis (200V, 25min) .</td>
<td>Two lanes: one lane with normal strip of around 1600bp, another one with a strip of only 100bp——>we supposed that there were problems with primer HR-F/HR-R. Primers formed too many dimers.
</td>
</tr>
<tr>
<td>7. 28</td>
<td>Tao and Zheng
</td>
<td>"Culture collection: 5 tubes of △smf1.<br> PCR: Clone of OsNramp5 target section (Detailed information is in the <a href="project-PROTOCOL ">protocol</a> ). Agarose gel (1%)electrophoresis (200V, 25min)."
</td>
<td>There were too many dimers. We supposed something was wrong with primer HR-F/HR-R and planned to change primers L-F/L-R and HR-F/HR-R.
</td>
</tr>
<tr>
<td>8. 02</td>
<td>Tao and Liu
</td>
<td>"Homologous recombination (linearized pFL61+<i>OsNramp5</i>)..<br> Transformation of DH5α: pFL61-OsNramp5-HR. Bred at constant temperature of 37 ℃ for 12 h. "
</td>
<td></td>
</tr>
<tr>
<td rowspan="3">8. 03</td>
<td>Liu and Zheng
</td>
<td>Pick transformants in the liquid YPD medium, Bred in shaker at 200 rpm/min, 30 ℃ for 12 h.
</td>
<td></td>
</tr>
<tr>
<td rowspan="2">Tao </td>
<td>Plasmid Extraction: DH5α (pFL61-OsNramp5-HR).
</td>
<td></td>
</tr>
<tr>
<td>Product of PCR (7. 27) are sent to sequence by TsingKe.
</td>
<td>We got the result of sequencing on August, 4th. It was the same as the sequence（LOC43422859） in NCBI.
</td>
</tr>
<tr>
<td rowspan="2">8. 04</td>
<td>Zheng
</td>
<td>"Activate DH5α (pFL61 and pFL61-OsNramp5-HR). Three zone marking on the LB amp medium."
</td>
<td></td>
</tr>
<tr>
<td>Tao
</td>
<td>Design primers of plasmid linearization and target gene amplification: pFL61-HR-F/pFL61-HR-R；Os-HR-F/Os-HR-R；short-F/short-R.
</td>
<td></td>
</tr>
<tr>
<td>8. 05
</td>
<td>Lin, Geng. Liu and Tao
</td>
<td>"Pick DH5α (pFL61 and pFL61-OsNramp5-HR) into liquid LB medium. Bred in shaker at 220 rpm/min, 30 ℃ for 12 h. <br> Plasmid Extraction: DH5α (pFL61-OsNramp5-HR). <br> PCR: Linearization of plasmid (model is pF61, primers are pFL61-HR-F/pFL61-HR-R,
the same below)<br> PCR: Clone of OsNramp5 target section (pFL61-OsNramp5-HR).<br> Homologous recombination (linearized pFL61+<i>OsNramp5</i>)."
</td>
<td></td>
</tr>
<tr>
<td rowspan="2">8. 06
</td>
<td>Liu and Geng
</td>
<td>"Confect solid YPD medium of 300 mL.<br> PCR: linearizationp the plasmid pFL61.<br> Clone of OsNramp5 target gene section. <br> Homologous recombination (linearized pFL61+<i>OsNramp5</i>).<br> Transformation of DH5α: pFL61-OsNramp5-HR.<br> Pick transformants. Bred at constant temperature of 37 ℃ for 12 h. "
</td>
<td></td>
</tr>
<tr>
<td>Lin and Tao
</td>
<td>"Plasmid Extraction: DH5α (pFL61). <br> <i>△ycf1</i> are bred in liquid YPD medium , in shaker at 200 rpm/min, 30 ℃ for 24 h.<br> Transformation of <i>△ycf1</i> (Detailed information is in <a href="project-PROTOCOL ">protocol</a>,
the same below): pFL61, pFL61-OsNramp5 and pFL61-OsNramp5-HR."
</td>
<td></td>
</tr>
</table>
</div>
<br>
<br>
<div class="notebook_cell ">
<table border="1">
<tr>
<th colspan="4">Experiment log of 8. 07-8. 13
</th>
</tr>
<tr>
<th>Date</th>
<th>Member</th>
<th>Experiment</th>
<th>Result</th>
</tr>
<tr>
<td rowspan="3">8. 07
</td>
<td>Lin and Tao
</td>
<td>"Confect solid SD-Ura medium of 150 mL (detailed prescription is in the <a href="project-PROTOCOL ">protocol</a>).<br> Make 10 medium."
</td>
<td></td>
</tr>
<tr>
<td>Liu
</td>
<td>Pick transformant (pFL61-OsNramp5-HR, 8. 06) into liquid LB medium. Bred in shaker at 220 rpm/min, 37 ℃ for 12 h.
</td>
<td></td>
</tr>
<tr>
<td>Geng
</td>
<td>Confect solid YPD medium of 150 mL.<br> Make 9 medium. Take <i>△ycf1</i> (8. 06) to spread on two medium. <br> Culture collection: 5 tubes of <i>△ycf1</i>. "
</td>
<td>The density of colonies was too high and there was no single colony. We need to streak.
</td>
</tr>
<tr>
<td rowspan="2">8. 08</td>
<td>Liu and Tao
</td>
<td>"<i>△ycf1</i> is streaked on the medium.<br> PCR(detailed information is in the <a href="project-PROTOCOL ">protocol</a>): Divide OsNramp5 into two parts and clone them respectively, to get ready for error-prone PCR (model
is pFL61-OsNramp5, primers are short-F/Os-HR-F；Os-HR-R/short-R)."
</td>
<td>There was nonspecific amplification happening in strip of 800 bp . Strips in all parts were in light color. We supposed something was wrong with anealing temperature.
</td>
</tr>
<tr>
<td>Geng
</td>
<td> PCR: Set gradient of anealing temperature (63 ℃; 65 ℃; 68 ℃) to PCR (model is pFL61-OsNramp5, primers are short-F/Os-HR-F；Os-HR-R/short-R).
</td>
<td></td>
</tr>
<tr>
<td rowspan="3">8. 09
</td>
<td rowspan='2'>Geng
</td>
<td>Agarose gel (1%) electrophoresis (200 V, 25 min) (product of PCR on August 8th).
</td>
<td>Strip was still not clear, and was in light color; we supposed there was comlementary pairing between primer and other area except target section of reconbinant plasmid.
</td>
</tr>
<tr>
<td>"PCR: Divide OsNramp5 into two parts and clone them respectively (model is cloned target section of OsNramp5, primer is short-F/Os-HR-F；Os-HR-R/short-R). Agarose gel (1%) electrophoresis (200 V, 25 min)."
</td>
<td>Strip was still not clear, and was in light color. Phenomenon of dimer is serious. We supposed somrthing was wrong with primer , and planned to change primer.
</td>
</tr>
<tr>
<td>Tao
</td>
<td>Design new primers for gene amplification: newshort-F/newshort-R.
</td>
<td></td>
</tr>
<tr>
<td rowspan="3">8. 10
</td>
<td>Tao
</td>
<td>Confect TE(aq) and LiAc(aq) of 50 mL respectively. <br> Transformation of <i>△ycf1</i>: pFL61-OsNramp5."
</td>
<td></td>
</tr>
<tr>
<td>Lin
</td>
<td>"Confect solid SD-Ura medium of 150 mL .<br> Make 10 medium. "
</td>
<td></td>
</tr>
<tr>
<td>Chen
</td>
<td>Transformation of DH5α: Pcada (from iGEM kit).
</td>
<td></td>
</tr>
<tr>
<td rowspan="2">8. 11
</td>
<td>Zheng
</td>
<td>"PCR: Divide OsNramp5 into two parts and clone them respectively(model is cloned target section of OsNramp5, primers are short-F/Os-HR-F；newshort-F/Os-HR-R). <br> PCR: Clone of target OsNramp5 section(model is pFL61-OsNramp5, primers
are Os-HR-F/Os-HR-R). <br> Agarose gel (1%)electrophoresis (200 V, 25 min). "
</td>
<td>"Strip of 800 bp was not clear. There was nonspecific amplification happening in strip of 800 bp and we can't seperate them. We supposed there was comlementary pairing between primer and other area except target section of reconbinant
plasmid ；<br> Strip of 1600bp was clear and obvious. Therefore, we planned to use long-section primers Os-HR-F/Os-HR-R to finish our following experiment. "
</td>
</tr>
<tr>
<td>Chen
</td>
<td>Pick transformants (Pcada) (8.10) into liquid LB medium. Bred in shaker at 220 rpm/min, 37 ℃ for 12 h.
</td>
<td></td>
</tr>
<tr>
<td rowspan="4">8. 12
</td>
<td>Liu
</td>
<td>Confect liquid SD-Ura medium of 100 mL .
</td>
<td></td>
</tr>
<tr>
<td rowspan="3">Lin , Geng and Tao
</td>
<td>"ep-PCR (KALANG kit, detailed information is in the <a href="project-PROTOCOL ">protocol</a>): Batches no. 1 and 2, 12 tubes (mdoel is cloned target section of OsNramp5 , primers are Os-HR-F/Os-HR-R).<br> Agarose gel (1%)
electrophoresis (200V, 25min)."
</td>
<td>There were only two strips in light color of 1600bp. The other lanes didn't have any strips. Then, we built a library labeled as ep1.We failed to build library labeled as ep2.
</td>
</tr>
<tr>
<td>Homologous recombination (linearized pFL61+<i>OsNramp5-mut</i>) ——>pFL61-OsNramp5-mut plasmid.<br> Transformation of <i>△ycf1</i>: pFL61-OsNramp5-mut.
</td>
<td></td>
</tr>
<tr>
<td>ep-PCR: Batch no. 3, 4 tubes (mdoel is cloned target section of OsNramp5 , primers are Os-HR-F/Os-HR-R).
</td>
<td></td>
</tr>
<tr>
<td>Chen
</td>
<td>Plasmid Extraction: DH5α (Pcada) (8.11).
</td>
<td></td>
</tr>
<tr>
<td rowspan="4">8. 13
</td>
<td>Geng
</td>
<td>Agarose gel (1%) electrophoresis (the second batch of OsNramp5-mut on 8. 12).
</td>
<td>Two results of agarose gel (1%) electrophoresis were similar. We didn't get obvious strips. We supposed this was related to reaction conditions of errpr-prone PCR and planned to change the conditions. We failed to build library
labeled as ep3.
</td>
</tr>
<tr>
<td>Lin and Tao
</td>
<td>"Confect solid SD-Ura medium of 300 mL .<br> Make 19 medium. "
</td>
<td></td>
</tr>
<tr>
<td>Liu and Geng
</td>
<td>"ep-PCR: Batch no. 4, use undiluted model (236 ng/μL) to react (mdoel is cloned target section of OsNramp5 , primers are Os-HR-F/Os-HR-R).<br> Agarose gel (1%) electrophoresis (200 V, 25 min)."
</td>
<td>There was no obvious strips. We failed to build library labeled as ep4.
</td>
</tr>
<tr>
<td>Tao
</td>
<td>"Transformation of DH5α: pFL61-OsNramp5-HR.<br> PCR: linearizationp the plasmid FL61 (model is pFL61, primers are pFL61-HR-F/pFL61-HR-R)."
</td>
<td></td>
</tr>
</table>
</div>
<br>
<br>
<div class="notebook_cell ">
<table border="1">
<tr>
<th colspan="4">
Experiment log of 8. 14-8. 20
</th>
</tr>
<tr>
<th>Date</th>
<th>Member</th>
<th>Experiment</th>
<th>Result</th>
</tr>
<tr>
<td rowspan="3">8. 14
</td>
<td>Zhu
</td>
<td>Configurate agar culture-medium and liquid YPD medium for 300 mL each.
</td>
<td></td>
</tr>
<tr>
<td>Liu and Zheng
</td>
<td>"Pick transformants (8. 13) into liquid LB medium. Bred in shaker at 220 rpm/min, 37 ℃ for 12 h.<br> Plasmid: pFL61-OsNramp5-HR."
</td>
<td></td>
</tr>
<tr>
<td>Geng
</td>
<td><i>△ycf1</i> (pFL61-OsNramp5-HR) are bred in liquid YPD medium. Bred in shaker at 200 rpm/min, 30 ℃ for 12 h.<br> <i>△ycf1</i> (pFL61-OsNramp5-HR) are bred in liquid SD-Ura medium. Bred in shaker at 200 rpm/min, 30 ℃ for 24 h.<br> Agarose gel (1%) electrophoresis (200 V, 25 min) (OsNramp5-mut on 8. 13).
</td>
<td>There was no obvious strip. We supposde this was related to KALANG kit.
</td>
</tr>
<tr>
<td rowspan="6">8. 15
</td>
<td>Zheng
</td>
<td>PCR: Test the result of homologous recombination (model is pFL61-OsNramp5-HR, primers are Os-HR-F/Os-HR-R).
</td>
<td>Strip of 1600 bp was clear and obvious , which meant we have suceeded in homologous recombination.
</td>
</tr>
<tr>
<td>Tao
</td>
<td>"ep-PCR (GenStar kit , detailed information is in the <a href="project-PROTOCOL ">protocol</a>) : Batch no. 5, set gradient of anealing temperature (55. 0 ℃, 57. 7 ℃, 59. 5 ℃, 61. 4 ℃ 63. 2 ℃), to test if the result of agarose
gel (1%) electrophoresis (on August 14th) is related to KALANG kit (model is pFL61-OsNramp5-HR, primers are Os-HR-F/Os-HR-R).<br> Agarose gel (1%) electrophoresis (200 V, 25 min)."
</td>
<td>There were obvious strips in all lanes of 1600bp. The degree of dispersion decreases with higher temperature. We made sure there was somerthing wrong with KALANG kit. We suceeded in building library labeled as ep5.
</td>
</tr>
<tr>
<td>Geng</td>
<td>"Homologous recombination (linearized pFL61+<i>OsNramp5-mut</i>) ——>pFL61-OsNramp5-mut plasmid.<br> Transformation of <i>△ycf1</i>: pFL61-OsNramp5-mut."
</td>
<td></td>
</tr>
<tr>
<td>Liu
</td>
<td>Transformation of DH5α: pFL61-OsNramp5-mut.
</td>
<td></td>
</tr>
<tr>
<td>Tao
</td>
<td>Transformation of <i>△ycf1</i>: pFL61-OsNramp5-mut.
</td>
<td></td>
</tr>
<tr>
<td>Chen
</td>
<td>Design primers for every cadmium-responding components：CadR-F/CadR-R; Plasmid-Linear-F/Plasmid-Linear-R; Pcada-sense/Pcada-antisense.
</td>
<td></td>
</tr>
<tr>
<td rowspan="2">8. 16
</td>
<td>Liu
</td>
<td>"Pick transformants (8. 16) into liquid LB medium. Bred in shaker at 220 rpm/min, 37 ℃ for 12 h.<br> E.coli fluid is sent to be sequenced."
</td>
<td>We got the sequencing result on August 18th. The results showed no signal.
</td>
</tr>
<tr>
<td>Lin
</td>
<td>Bacteria Liquid PCR.
</td>
<td>Strip of 1600 bp was clear and obvious , which meant we have suceeded in homologous recombination.
</td>
</tr>
<tr>
<td rowspan="2">8. 17
</td>
<td>Geng
</td>
<td>ep-PCR: Batch no. 5, 12 tubes (mdoel is cloned target section of OsNramp5 , primers are Os-HR-F/Os-HR-R).
</td>
<td></td>
</tr>
<tr>
<td>Liu
</td>
<td>"Homologous recombination (linearized pFL61+<i>OsNramp5-mut</i>). Transformation of <i>△ycf1</i>: pFL61-OsNramp5-mut."
</td>
<td></td>
</tr>
<tr>
<td rowspan="2">8. 18
</td>
<td>Lin
</td>
<td>Configurate agar culture-medium for 400 mL and liquid YPD medium for 150 mL .
</td>
<td></td>
</tr>
<tr>
<td>Chen
</td>
<td>"PCR: Clone of gene of <i>gfp</i> (model is plasmid K608011，primers are Plasmid-Linear-F/Plasmid-Linear-R).<br> Pick <i>Pseudomonas aeruginosa</i> into liquid LB medium. Bred in shaker at 200 rpm/min, 37 ℃ for 12 h."
</td>
<td>The liquid medium is not cloudy (8.19).
</td>
</tr>
<tr>
<td rowspan="5">8. 19
</td>
<td>Zheng
</td>
<td>"ep-PCR: Batch no. 6, 5 tubes (mdoel is cloned target section of OsNramp5 , primers are Os-HR-F/Os-HR-R).<br> Homologous recombination (linearized pFL61+<i>OsNramp5-mut</i>)."
</td>
<td>The degree of dispersion of strips was serious, which meant we failed to build the library labeled as ep6.
</td>
</tr>
<tr>
<td>Liu
</td>
<td>Plasmid (8. 16) Extraction: pFL61-OsNramp5-mut.
</td>
<td></td>
</tr>
<tr>
<td>Geng
</td>
<td>Agarose gel (1%) electrophoresis (200 V, 25 min) (pFL61-OsNramp5-mut and pFL61).
</td>
<td>pFL61-OsNramp5-mut showed clear and obvious stips in the area of 7000 bp, and pFL61 showed clear and obvious stips in the area of 5000 bp, which meant we have suceeded in homologous recombination.
</td>
</tr>
<tr>
<td>Tao
</td>
<td>"Transformation of <i>△ycf1</i>: pFL61-OsNramp5-mut. Pick 7 transformants that grow well into liquid SD-Ura medium. Bred in shaker at 200 rpm/min, 30℃ for 24 h . Label as ep1-1 - ep1-7."
</td>
<td></td>
</tr>
<tr>
<td>Chen
</td>
<td>"Pick Pseudomonas aeruginosa into liquid LB medium. Bred in shaker at 200 rpm/min, 37 ℃ for 12 h.<br> Bacterial fluid PCR: Clone of <i>cadR</i>> (take one of the single colony on the medium into PCR system as model，primers are
CadR-F/CadR-R).
<br>
<i>PcadA</i> obtaining：DNA double strands are obtained by annealing of two single strands (Pcada-sense/Pcada-antisense)<br> Homologous recombination (<i>gfp</i>+<i>PcadA</i>>+<i>cadR</i>+PSB1C3). Transformation of DH5α: cadR-PcadA-GFP."
</td>
<td></td>
</tr>
<tr>
<td rowspan="3">8. 20
</td>
<td>Tao and Lin
</td>
<td>"Confect solid SD-Ura (Cd, 30 μM) medium of 150 mL.<br> <i>△ycf1</i> (pFL61 and pFL61-OsNramp5-HR) are bred in liquid SD-Ura medium. Bred in shaker at 200 rpm/min , 30 ℃ for 24 h. "
</td>
<td>After 4 days, there was no transformants growing on the medium, which meant we failed to build library labeled as ep7.
</td>
</tr>
<tr>
<td>Geng and Liu
</td>
<td>ep-PCR: Batch no. 7, 4 tubes (mdoel is cloned target section of OsNramp5 , primers are Os-HR-F/Os-HR-R, anealing temperature is set as 60 ℃).
</td>
<td></td>
</tr>
<tr>
<td>Chen
</td>
<td>Pick transformants (cadR-PcadA-GFP) (8.19) into liquid LB medium. Bred in shaker at 220 rpm/min, 37 ℃ for 12 h.
</td>
<td></td>
</tr>
</table>
</div>
<br>
<br>
<div class="notebook_cell ">
<table border="1">
<tr>
<th colspan="4">
Experiment log of 8. 21-8. 26
</th>
</tr>
<tr>
<td>Date</td>
<td>Member</td>
<td>Experiment</td>
<td>Result</td>
</tr>
<tr>
<td rowspan="3">8. 21
</td>
<td>Zheng</td>
<td>PCR: Linearizationp the plasmid pFL61.
</td>
<td>No strips, which meant we failed to linearize the plasmid, and we planned to do it again.
</td>
</tr>
<tr>
<td>Lin
</td>
<td>Pick transformants. Streak on SD-Ura medium to build library (ep5).
</td>
<td></td>
</tr>
<tr>
<td>Chen
</td>
<td>"Culture collection: DH5α (cadR-PcadA-GFP) (8.20).<br> 5 tubes of DH5α (cadR-PcadA-GFP) fluid are sent to be sequenced."
</td>
<td>We got the result of sequencing on August 22th. One of four tubes of bacteria fluid is plasmid that were reconbinated successfully.
</td>
</tr>
<tr>
<td rowspan="2">8. 22
</td>
<td>Zheng
</td>
<td>PCR: Linearizationp the plasmid FL61.
</td>
<td>No strips, which meant we failed to linearize the plasmid, and we planned to do it again.
</td>
</tr>
<tr>
<td>Zhu
</td>
<td>Confect solid SD-Ura medium of 800 mL .
</td>
<td></td>
</tr>
<tr>
<td rowspan="6">8. 23
</td>
<td>Liu
</td>
<td>"Confect liquid SD-Ura medium of 300 mL .<br> PCR: Linearizationp the plasmid pFL61.<br> Agarose gel (1%) electrophoresis (200 V, 25 min)."
</td>
<td>There showed strips, which meant we suceeded in plamid linearization. We supposed there was something with pF61 model.
</td>
</tr>
<tr>
<td>Zhu
</td>
<td>"<i>△ycf1</i> are bred in liquid YPD mediumBred in shaker at 200 rpm/min, 30 ℃ for 12 h.<br> Streak on the medium. "
</td>
<td></td>
</tr>
<tr>
<td>Tao
</td>
<td>"DH5α (pFL61) are bred in liquid LB medium. Bred at 220 rpm/min, 37 ℃ for 12 h.<br> Streak on LB amp medium."
</td>
<td></td>
</tr>
<tr>
<td>Zheng
</td>
<td>Agarose gel (1%) electrophoresis (200 V, 25 min) (pFL61).
</td>
<td>There was no obvious strips. We made sure pF61 have degraded.
</td>
</tr>
<tr>
<td>Lin
</td>
<td>Measure OD<sub>600</sub> of <i>△ycf1</i> (ep1-1 - ep1-7 and pFL62, pFL61-OsNramp5-HR).
</td>
<td>Maximum of OD<sub>600</sub> was around 0. 4. We supposed the time of yeast fluid shkaing was too short.
</td>
</tr>
<tr>
<td>Chen
</td>
<td>Plasmid (8.20) Extraction: DH5α (cadR-PcadA-GFP).
</td>
<td></td>
</tr>
<tr>
<td rowspan="3">8. 24
</td>
<td>Liu
</td>
<td>Plasmid (8.23) Extraction: pFL61.
</td>
<td></td>
</tr>
<tr>
<td>Tao
</td>
<td>"ep-PCR: Batch no. 8, 5 tubes (mdoel is cloned target section of OsNramp5 , primers are Os-HR-F/Os-HR-R).<br> Homologous recombination (plasmid linearization of pFL61+<i>OsNramp5-mut</i>). <br> Transformation of <i>△ycf1</i>: pFL61-OsNramp5-mut.<br> <i>△ycf1</i> (pFL61, pFL61-OsNramp5-HR and ep5-1 - ep5-8) are bred in liqiuid SD-Ura medium. Bred at 200 rpm/min, 30 ℃ for 24 h."
</td>
<td>After 4 days, there was no transformants growing on the medium, which meant we fail to build library labeled as ep8.
</td>
</tr>
<tr>
<td>Chen
</td>
<td>Transformation of BL21: cadR-PcadA-GFP.
</td>
<td></td>
</tr>
<tr>
<td rowspan="4">8. 25
</td>
<td>Zheng
</td>
<td>PCR: Linearization of the plasmid pFL61.
</td>
<td>Strips were not clear.There was nonspecific amplification happening. We supposed the concentration of reaction model was too high.
</td>
</tr>
<tr>
<td>Liu
</td>
<td>ep-PCR: Batch no. 8, 5 tubes (mdoel is cloned target section of OsNramp5 , primers are Os-HR-F/Os-HR-R, enhancer in the reaction system is added to 10 μL).
</td>
<td></td>
</tr>
<tr>
<td>Tao
</td>
<td>Measure OD<sub>600</sub> of <i>△ycf1</i> (ep5-1 - ep5-8 and pFL62, pFL61-OsNramp5-HR).
</td>
<td>Maximum of OD<sub>600</sub> was around 0. 6.
</td>
</tr>
<tr>
<td>Chen
</td>
<td>Characterization pre-experiment of cadmium ion concentration responding biosenser.
</td>
<td>Detailed information is in the <a href="results#download">Supplementary Information-2</a>.
</td>
</tr>
<tr>
<td rowspan="4">8. 26
</td>
<td>Zheng
</td>
<td>PCR: Linearization of the plasmid pFL61 (change the PCR equipment).
</td>
<td>Strips were clear and obvious. We supposed the bad results of plasmid linearization was caused by PCR amplifier.
</td>
</tr>
<tr>
<td>Tao
</td>
<td>"Homologous recombination (linearized pFL61+<i>OsNramp5</i> or <i>OsNramp5-mut</i>).<br> Transformation of <i>△ycf1</i>: pFL61-OsNramp5-HR or pFL61-OsNramp5-mut.<br> Measure OD<sub>600</sub> of <i>△ycf1</i> (ep5-1 - ep5-8 and pFL62,
pFL61-OsNramp5-HR)."
</td>
<td>OD<sub>600</sub> were all over 0. 6.
</td>
</tr>
<tr>
<td>Lin
</td>
<td>Spot (detailed information is in the <a href="project-PROTOCOL ">protocol</a>).
</td>
<td></td>
</tr>
<tr>
<td>Chen
</td>
<td>Characterization experiment of cadmium ion concentration responding biosenser.
</td>
<td>Detailed information is in the <a href="results#download ">Supplementary Information-2</a>.
</td>
</tr>
</table>
</div>
<br>
<br>
<div class="notebook_cell ">
<table border="1">
<tr>
<th colspan="3">Experiment log of 8.26-9.25
</th>
</tr>
<tr>
<td rowspan="3">8.26-9.25
</td>
<td>Geng
</td>
<td>ep-PCR (mdoel is cloned target section of OsNramp5, primers are Os-HR-F/Os-HR-R), five batches in total. Twenty tubes of homologous recombination (linearized pFL61+<i>OsNramp5-mut</i>).
</td>
</tr>
<tr>
<td>Liu and Zhu
</td>
<td>"Transformation of <i>△ycf1</i>：pFL61-OsNramp5-HR or pFL61-OsNramp5-mut . <br> Pick transformants, streak on the SD-Ura medium to build library (ep4-1 - ep3‘-33) (detailed timetable is as following)."
</td>
</tr>
<tr>
<td>Lin, Geng, Liu and Tao
</td>
<td><i>△ycf1</i> (pFL61, pFL61-OsNramp5-HR and pFL61-OsNramp5-mut) are bred in liquid SD-Ura medium. Bred in shaker at 200 rpm/min, 30 ℃ for 24 h . Measure OD<sub>600</sub> of <i>△ycf1</i> (pFL61, pFL61-OsNramp5-HR and pFL61-OsNramp5-mut).
Spot (detailed timetable is as following)."
</td>
</tr>
</table>
</div>
<br>
<br>
<div class="notebook_cell ">
<table border='1'>
<tr>
<th colspan="3">Timetable</th>
</tr>
<tr>
<th>Number</th>
<th>Time of Culturing</th>
<th>Time of Spotting</th>
</tr>
<tr>
<td>ep4-1</td>
<td class='x25'>8.27 a.m.</td>
<td class='x27'>8.28 p.m.</td>
</tr>
<tr class='x32'>
<td>ep4-2</td>
<td class='x25'>8.27 a.m.</td>
<td class='x27'>8.28 p.m.</td>
</tr>
<tr>
<td>ep5-1</td>
<td class='x25'>8.24 p.m.</td>
<td class='x27'>8.26 p.m.</td>
</tr>
<tr>
<td>ep5-2</td>
<td class='x25'>8.24 p.m.</td>
<td class='x27'>8.26 p.m.</td>
</tr>
<tr>
<td>ep5-3</td>
<td class='x25'>8.24 p.m.</td>
<td class='x27'>8.26 p.m.</td>
</tr>
<tr>
<td>ep5-4</td>
<td class='x25'>8.24 p.m.</td>
<td class='x27'>8.26 p.m.</td>
</tr>
<tr>
<td>ep5-5</td>
<td class='x25'>8.24 p.m.</td>
<td class='x27'>8.26 p.m.</td>
</tr>
<tr>
<td>ep5-6</td>
<td class='x25'>8.24 p.m.</td>
<td class='x27'>8.26 p.m.</td>
</tr>
<tr>
<td>ep5-7</td>
<td class='x25'>8.24 p.m.</td>
<td class='x27'>8.26 p.m.</td>
</tr>
<tr>
<td>ep5-8</td>
<td class='x25'>8.24 p.m.</td>
<td class='x27'>8.26 p.m.</td>
</tr>