Difference between revisions of "Team:CityU HK/Notebook"
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</tr> | </tr> | ||
</table> | </table> | ||
+ | |||
+ | <table id="t01" border="1"> | ||
+ | <tr> | ||
+ | <th colspan="5">Week 8 : July 6 – July 10</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="5"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="20%">Date</td> | ||
+ | <td width="20%">Group 1</td> | ||
+ | <td width="20%">Group 2</td> | ||
+ | <td width="20%">Group 3</td> | ||
+ | <td width="20%">Group 4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Monday 6</td> | ||
+ | <td><li>Gel purification of the [X/P] digested pSB1C3 vector isolated on July 3</li> <li><font color="#D633FF">PCR amplification of the BBa_K124017 Rzλ(ori) gene using a F-primer with SacII restriction site</font></li></td> | ||
+ | <td></td> | ||
+ | <td><li><font color="#00CC00">Transformation of the ligation product pSB1C3-Rλ(co)-Rzλ(ori) (from July 3 [3])</font></li></td> | ||
+ | <td><li>Restriction digestion of July 3 Rλ(co) with [S/P]</li><li>Gel purification</li> <br> <li>Restriction digestion of Rzλ(co) (from Gp2 June 29) with [X/P]</li><li>Gel purification</li></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Tuesday 7</td> | ||
+ | <td><li><font color="#D633FF">Gel electrophoresis of the SacII digested Rzλ(ori) PCR amplicon</font></li> <li><font color="#0099FF">PCR amplification of SλWT(ori) using a new primer pair</font></li></td> | ||
+ | <td><li><font color="#0099FF">Extraction of the plasmid pGOv4-SλWT(co)</font></li><li><font color="#D633FF">Restriction digestion with [X] on July 3 [P] digested Rλ(ori)</font></li></td> | ||
+ | <td><li><font color="#00CC00">Colony PCR on July 6 Rλ(co)-Rzλ(ori) clones showed no bands</li> <li>Redo July 3’s ligation of [X/P] digested Rzλ(ori) insert into [S/P] digested pSB1C3-Rλ(co) vector</li> <li>Transformation</font></li></td> | ||
+ | <td><li>Ligation of the [X/P] digested Rzλ(co) insert into the [S/P] digested pSB1C3-Rλ(co) vector</li> <li>Transformation</li></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Wednesday 8</td> | ||
+ | <td><li><font color="#D633FF">PCR purification of July 6 SacII Rzλ(ori) PCR product</font></li></td> | ||
+ | <td><li><font color="#D633FF">Ligation of [X/P] digested Rλ(ori) into Gp1 July 6 [X/P]</li><li>Transformation of 7/7’s ligation product Rλ(ori) in pSB1C3</font></li></td> | ||
+ | <td><li><font color="#00CC00">Colony PCR on July 7 Rλ(co)-Rzλ(ori) clones still showed no bands</font></li></td> | ||
+ | <td><li>Colony PCR on July 7 Rλ(co)-Rzλ(co) clones showed no bands</li></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Thursday 9</td> | ||
+ | <td><li><font color="#D633FF">Restriction digestion of SacII Rzλ(ori) with [SacII/P]</font></li><li><font color="#00CC00">Restriction digestion of Gp2 July 7 SλWT(co) with [E/S]</font></li><li><font color="#0099FF">Gel electrophoresis of July 7 SλWT(ori)</li><li>PCR purification of SλWT(ori)</font></li></td> | ||
+ | <td><li><font color="#D633FF">Colony PCR on Rλ(ori) (from July 8 transformed cells)</font></li> <li><font color="#0099FF">Restriction digestion of July 7 SλWT(co) with [E/P]</font></li></td> | ||
+ | <td><li><font color="#D633FF">Ligation of [X/P] July 3 digested Rzλ(ori) insert into [X/P] digested pSB1C3 vector</li> <li>Transformation</font></li></td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Friday 10</td> | ||
+ | <td><li>Restriction digestion of SλWT(ori) with [X/P] & Gel purification</li><li>Ligation of [X/P] digested SλWT(ori) insert into July 6 [X/P] digested pSB1C3 vector</li><li>Transformation</li><li>Gel purification of July 9 [E/S] digested SλWT(co)</li></td> | ||
+ | <td><li><font color="#0099FF">Gel purification of July 9 [E/P] digested SλWT(co)</font></li></td> | ||
+ | <td><li><font color="#D633FF">Colony PCR on July 9 Rzλ(ori) clones showed no bands</font></li><li><font color="#00CC00">Restriction analysis on July 7 transformed cells</li><li>Another redo of the ligation of July 3 [X/P] digested Rzλ(ori) insert into July 3 [S/P] digested pSB1C3-Rλ(co)</li><li>Transformation</font></li></td> | ||
+ | <td><li>Extraction of the plasmid pSB1C3-Rλ(co)-Rzλ(co) (from July 7 transformed cells)</li> <li>Restriction analysis of Rλ(co)-Rzλ(co) showed bands of the expected size</li></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <table id="t01" border="1"> | ||
+ | <tr> | ||
+ | <th colspan="5">Week 9 : July 13 – July 18</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="5"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="20%">Date</td> | ||
+ | <td width="20%">Group 1</td> | ||
+ | <td width="20%">Group 2</td> | ||
+ | <td width="20%">Group 3</td> | ||
+ | <td width="20%">Group 4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Monday 13</td> | ||
+ | <td><li><font color="#0099FF">Colony PCR on SλWT(ori) clones showed bands of the expected size</font></li><li><font color="#00CC00">Ligation of [E/S] digested SλWT(co) insert into pSB1C3 vector</li><li>Transformation</font></li></td> | ||
+ | <td><li><font color="#D633FF">Extraction of the plasmid pSB1C3-Rλ(ori)</li><li>Restriction digestion with [X/P]</li><li>Gel purification </font></li></td> | ||
+ | <td><li><font color="#00CC00">Colony PCR on 10/7 transformed cells pSB1C3-Rλ(co)-Rzλ(ori)</font></li></td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Tuesday 14</td> | ||
+ | <td><li><font color="#00CC00">Colony PCR on SλWT(co) clones showed bands of the expected size</font></li> <li><font color="#FF3333"> Restriction digestion of Gp2 Rλ(ori) with [X/SacII]</font></li></td> | ||
+ | <td><li><font color="#D633FF">Restriction analysis of [X/P] digested Rλ(ori)</font></li><li>Restriction digestion of Rzλ(co) with [X/P]</li><li>Gel purification</li></td> | ||
+ | <td></td> | ||
+ | <td><li>Extraction of the plasmid pSB1C3-Rλ(co)-Rzλ(co)</li> <li>Restriction digestion with [X/P]</li></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Wednesday 15</td> | ||
+ | <td><li><font color="#FF3333">Gel purification of July 14 [X/SacII] digested Rλ(ori)</font></li><br><li><font color="#FF9900">Ligation between</font> <font color="#00CC00">[E/S] digested SλWT(co)</font>, <font color="#FF3333">[X/SacII] digested Rλ(ori)</font>, <font color="#D633FF">[SacII/P] digested Rzλ(ori)</font> and <font color="#FF9900">[E/P] digested pSB1C3 backbone</li><li>Transformation</font></li></td> | ||
+ | <td><li><font color="#D633FF">Extraction of the plasmid pSB1C3-Rλ(ori)</li><li>Restriction digestion with [S/P]</font></li></td> | ||
+ | <td><li>Extraction of the plasmid pSB1C3-Rλ(co)</li><li>Restriction digestion of Rλ(co) with [E/S]</li><li>Restriction digestion of [E/X] digested Rzλ(ori) with CIP & Gel purification</li><li>Ligation of the [E/S] digested Rλ(co) insert into [E/X] digested pSB1C3-Rzλ(ori)</li><li>Transformation</li></td> | ||
+ | <td><li>Gel purification of July 14 [X/P] digested Rλ(co)-Rzλ(co)</li></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Thursday 16</td> | ||
+ | <td><li><font color="#FF9900">Colony PCR on SλWT(co)- Rλ(ori)-Rzλ(ori) showed bands believed to be the desired insert</font></li></td> | ||
+ | <td><li><font color="#D633FF">Gel purification of July 15 [S/P] digested Rλ(ori)</font></li> <li>Ligation of the [X/P] digested Rzλ(co) insert into <font color="#D633FF">[S/P] digestd pSB1C3-Rλ(ori) vector</font></li></td> | ||
+ | <td><li>Colony PCR on July 15 transformed cells</li> <li>Redo the ligation of [E/S] digested Rλ(co) insert into [E/X] digested pSB1C3-Rzλ(ori)</li> <li>Transformation</li></td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Friday 17</td> | ||
+ | <td></td> | ||
+ | <td><li>Transformation of July 16 ligation product pSB1C3-Rλ(ori)-Rzλ(co)</li></td> | ||
+ | <td><li>Colony PCR on 16/7 transformed cells</li><li>Restriction digestion of Sλ(co) with [E/S] & Gel purification</li><li>Redo the ligation of [E/S] digested Rλ(co) insert into [E/X] digested pSB1C3-Rzλ(ori)</li><li>Transformation</li></td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Saturday 18</td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td><li><font color="#D633FF">Extraction of the plasmids pSB1C3-Sλmut(ori) and pSB1C3-Sλmut(co)</li><li>Restriction digestion with [S/P]</font></li></td> | ||
+ | </table> | ||
+ | |||
+ | <br> | ||
+ | <p>DNA sequencing results later revealed that the genes amplified from the BioBrick, BBa_K124017, did not contain the desired sequences. Genomic DNA from E. coli BL21(DE3) will be used as template (starting from week 10) to create new λ(ori) related BioBricks. Gene sequences of the λ(co) clones were correct.</p> | ||
+ | <br> | ||
+ | |||
+ | <table id="t01" border="1"> | ||
+ | <tr> | ||
+ | <th colspan="5">Week 10 : July 20 – July 25</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="5"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="20%">Date</td> | ||
+ | <td width="20%">Group 1</td> | ||
+ | <td width="20%">Group 2</td> | ||
+ | <td width="20%">Group 3</td> | ||
+ | <td width="20%">Group 4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Monday 20</td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td><li><font color="#D633FF"> Gel purification of July 18 [S/P] digested pSB1C3-Sλmut(ori) and pSB1C3-Sλmut(co)</font></li><li><font color="#0099FF">Ligation of July 15 [X/P] digested Rλ(co)-Rzλ(co) insert into the [S/P] digested pSB1C3-Sλmut(ori) vector</font></li><li><font color="#FF9900"> Ligation of July 15 [X/P] digested Rλ(co)-Rzλ(co) insert into the [S/P] digested pSB1C3-Sλmut(co) vector</font></li></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Tuesday 21</td> | ||
+ | <td></td> | ||
+ | <td><li>Transformation of pGOv4-Ribo_Sλ(co), pGOv4-Ribo_Rλ(co) and pGOv4-Ribo_Rzλ(co) into competent <i>E. coli</i> cells</li><li>Transformation of pGOv4-SλWT(co)-Rλ(co)-Rzλ(co) (λLysis(c_c_c))</li></td> | ||
+ | <td></td> | ||
+ | <td><li><font color="#0099FF">Transformation of the pSB1C3-Sλmut(ori)-Rλ(co)-Rzλ(co) (λLysis_Sm(o_c_c))</font></li> <li><font color="#FF9900"> Transformation of the pSB1C3-Sλmut(co)-Rλ(co)-Rzλ(co) (λLysis_Sm(c_c_c))</font></li></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Wednesday 22</td> | ||
+ | <td><li><font color=”#3333FF”>Genomic DNA extraction of BL21(DE3) <i>E. coli</i> cells</li><li>PCR amplification of the Sλ(ori), Rλ(ori) and Rzλ(ori) genes using the extracted genomic DNA as template</font></li></td> | ||
+ | <td></td> | ||
+ | <td><li><font color="#FF00FF">Redo Gp1 July 21 transformation of the pGOv4-Ribo_Sλ(co) and pGOv4-SλWT(co)-Rλ(co)-Rzλ(co) (λLysis (c_c_c)) into competent <i>E. coli</i> cells</font></li></td> | ||
+ | <td><li><font color="#0099FF"> Colony PCR on λLysis_Sm(o_c_c) clones showed bands of the expected size</font></li> <br> <li><font color="#FF9900">Colony PCR on c clones revealed that that the colonies contained inserts of the wrong sizes</li><li>Redo the restriction digestion of Rλ(co)-Rzλ(co) with [X/P] & Gel purification</font></li></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Thursday 23</td> | ||
+ | <td><li><font color="#FF00FF">Extraction of the plasmids pGOv4-Ribo_Sλ(co), pGOv4-Ribo_Rλ(co) and pGOv4-Ribo_Rzλ(co)</li><li>Restriction digestion of the extracted plasmids with [E/P]</li><li>Gel purification</font></li></td> | ||
+ | <td></td> | ||
+ | <td><li><font color="#FF00FF">Extraction of the plasmid pGOv4-λLysis(c_c_c)</li> <li>Restriction digestion with [E/P]</font></li></td> | ||
+ | <td><li><font color="#FF9900">Redo the ligation of the [X/P] digested Rλ(co)-Rzλ(co) insert into the [S/P] digested pSB1C3-Sλmut(co) vector</li><li>Transformation</font></li></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Friday 24</td> | ||
+ | <td><li><font color="#FF00FF">Ligation of the [E/P] digested Ribo_Sλ(co) and Ribo_Sλ(co) inserts into [E/P] digested pSB1C3 respectively</li><li>Transformation</font></li><br><li><font color="#3333FF">Restriction digestion of the Sλ(ori), Rλ(ori) and Rzλ(ori) amplicons with [X/P]</font></li></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td><li><font color="#FF9900">Colony PCR on the new λLysis_Sm(c_c_c) clones showed bands of the expected size. However, later sequencing result revealed that the colony was actually a mixed one with both the correct plasmid and the self-ligated RRz(co) plasmid</font></li></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Saturday 25</td> | ||
+ | <td><li><font color="#FF00FF">Colony PCR on July 24 transformed Ribo_Sλ and Ribo_Rλ clones</font></li><br><li><font color="#3333FF">Ligation of the [X/P] digested Sλ(ori), Rλ(ori) and Rzλ(ori) inserts into [X/P] digested pSB1C3 respectively</li><li>Transformation</font></li></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | </table> | ||
+ | |||
Revision as of 18:27, 19 August 2015
Keys to the table:
[?/?]: Names of restriction enzyme used for digestion:; E, EcoRI; X, XbaI; S, SpeI; P, PstI
- A ribosome binding site (RBS) is linked upstream to each gene
- The host cells used for transformation were competent JM109 E. coli cells
PCR amplification of the lacZ and lacY genes.
Week 1 : May 18 – 22 | ||||
---|---|---|---|---|
Date | Group 1 | Group 2 | Group 3 | Group 4 |
Monday 18 | ||||
Tuesday 19 | ||||
Wednesday 20 | ||||
Thursday 21 | ||||
Friday 22 |
Week 2 : May 25 – 29 | ||||
---|---|---|---|---|
Date | Group 1 | Group 2 | Group 3 | Group 4 |
Monday 25 | ||||
Tuesday 26 | ||||
Wednesday 27 | ||||
Thursday 28 | ||||
Friday 29 |
Groups 1 & 3 and Groups 2 & 4 used different cloning strategies to assemble the biobrick J23100_lacZ-lacY.
Week 3 : June 1 – 5 | ||||
---|---|---|---|---|
Date | Group 1 | Group 3 | Group 2 | Group 4 |
Monday 1 | ||||
Tuesday 2 | ||||
Wednesday 3 | ||||
Thursday 4 | ||||
Friday 5 |
Week 4 : June 8 – 12 | ||||
---|---|---|---|---|
Date | Group 1 | Group 3 | Group 2 | Group 4 |
Monday 8 | ||||
Tuesday 9 | ||||
Wednesday 10 |
Week 8 : July 6 – 10 | ||
---|---|---|
Date | Remarks | |
Monday 6 | ||
Tuesday 7 | Not successful | |
Wednesday 8 | Not successful | |
Thursday 9 | ||
Friday 10 | Not successful |
Week 9 : July 13 – 18 | ||
---|---|---|
Date | Remarks | |
Monday 13 | Successful | |
Tuesday 14 | Successful | |
Wednesday 15 | Successful | |
Thursday 16 | Successful | |
Friday 17 | ||
Saturday 18 | Successful |
Week 10 : July 20 – 25 | ||
---|---|---|
Date | Remarks | |
Monday 20 | Successful | |
Tuesday 21 | ||
Wednesday 22 | ||
Thursday 23 | ||
Friday 24 |
Keys to the table:
ori: original gene sequence, without codon optimization
co: codon optimized
λ Lysis(o_o_o): SλWT(ori)-Rλ(ori)-Rzλ(ori)
λLysis(c_c_c): SλWT(co)-Rλ(co)-Rzλ(co)
λLysis_Sm(o_c_c): Sλmut(ori)-Rλ(co)-Rzλ(co)
λLysis_Sm(c_c_c): Sλmut(co)-Rλ(co)-Rzλ(co)
[x/y]: restriction sites digested: E, EcoRI; X, XbaI; S, SpeI; P, PstI
Gene name or plasmid name [x/y]: the gene fragment or plasmid flanked with the restriction sites x and y at the ends
-A ribosome binding site (RBS) is linked upstream of each gene
-Coloring refers to sub-tasks in that particular group
-The cells used for transformation were competent JM109 E. coli cells
Week 4 : June 8 – 12 | ||||
---|---|---|---|---|
Date | Group 1 | Group 2 | Group 3 | Group 4 |
Monday 8 | ||||
Tuesday 9 | ||||
Wednesday 10 | ||||
Thursday 11 | ||||
Friday 12 |
Week 5 : June 15 – 19 | ||||
---|---|---|---|---|
Date | Group 1 | Group 2 | Group 3 | Group 4 |
Monday 15 | ||||
Tuesday 16 | ||||
Wednesday 17 | ||||
Thursday 18 | ||||
Friday 19 |
Week 6 : June 22 – 26 | ||||
---|---|---|---|---|
Date | Group 1 | Group 2 | Group 3 | Group 4 |
Monday 22 | ||||
Tuesday 23 | ||||
Wednesday 24 | ||||
Thursday 25 | ||||
Friday 26 |
Week 7 : June 29 – July 3 | ||||
---|---|---|---|---|
Date | Group 1 | Group 2 | Group 3 | Group 4 |
Monday 29 | ||||
Tuesday 30 | ||||
Wednesday 1 | ||||
Thursday 2 | ||||
Friday 3 | [1] |
Week 8 : July 6 – July 10 | ||||
---|---|---|---|---|
Date | Group 1 | Group 2 | Group 3 | Group 4 |
Monday 6 | ||||
Tuesday 7 | ||||
Wednesday 8 | ||||
Thursday 9 | ||||
Friday 10 |
Week 9 : July 13 – July 18 | ||||
---|---|---|---|---|
Date | Group 1 | Group 2 | Group 3 | Group 4 |
Monday 13 | ||||
Tuesday 14 | ||||
Wednesday 15 | ||||
Thursday 16 | ||||
Friday 17 | ||||
Saturday 18 |
DNA sequencing results later revealed that the genes amplified from the BioBrick, BBa_K124017, did not contain the desired sequences. Genomic DNA from E. coli BL21(DE3) will be used as template (starting from week 10) to create new λ(ori) related BioBricks. Gene sequences of the λ(co) clones were correct.
Week 10 : July 20 – July 25 | ||||
---|---|---|---|---|
Date | Group 1 | Group 2 | Group 3 | Group 4 |
Monday 20 | ||||
Tuesday 21 | ||||
Wednesday 22 | ||||
Thursday 23 | ||||
Friday 24 | ||||
Saturday 25 |