Difference between revisions of "Team:CityU HK/Notebook/Lysis plasmid (phage 21)"

Revision as of 07:57, 10 September 2015

Notebook

Lysis plasmid (phage 21)

Keys to the table:
ori: original gene sequence, without codon optimization
co: codon optimized
21Lysis(o_o_o): S21WT(ori)-Rλ(ori)-Rzλ(ori)
21Lysis(c_c_c): S21WT(co)-Rλ(co)-Rzλ(co)
21Lysis_Sm(o_c_c): S21mut(ori)-Rλ(co)-Rzλ(co)
21Lysis_Sm(c_c_c): S21mut(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				Extraction of the plasmid pGOv4-Rz21(co) by mini-prep Restriction digestion with [E/P]
Thursday 11				Gel purification of the June 10 [E/P] digested Rz21(co) Ligation of the [E/P] digested Rz21(co) insert into [E/P] digested pSB1C3 Transformation
Friday 12				Colony PCR on Rz21(co) clones showed bands of the expected size

Week 5 : June 15 – 19
Date	Group 1	Group 2	Group 3	Group 4
Monday 15
Tuesday 16	Plasmid extraction of the pGOv4-R21(co) Restriction digestion with [E/P]
Wednesday 17	Gel purification of the [E/P] digested R21(co) Ligation of the [E/P] digested R21(co) insert into the [E/P] digested pSB1C3 Transformation
Thursday 18	Colony PCR confirmed the presence of the R21(co) insert
Friday 19

Week 6 : June 22 – 26
Date	Group 1	Group 2	Group 3	Group 4
Monday 22	Extraction of June 17 plasmid pSB1C3-R21(co)
Tuesday 23				Extraction of June 11 plasmid pSB1C3-Rz21(co) Restriction analysis with [E/P]
Wednesday 24
Thursday 25
Friday 26			PCR amplification on phage 21 wild type lysis cassette

Week 7 : June 29 – July 3
Date	Group 1	Group 2	Group 3	Group 4
Monday 29	Restriction digestion of June 22 R21(co) with [S/P] & Gel purification			Extraction of June 11 plasmid pSB1C3-Rz21(co) Restriction digestion with [X/P]
Tuesday 30			Redo the PCR amplification on phage 21 wild type lysis cassette	Redo June 29’s restriction digestion of Rz21(co) with [X/P]
Wednesday 1
Thursday 2				Gel purification of [X/P] digested Rz21(co) Ligation of the [X/P] digested Rz21(co) insert into Gp1 June 29 [S/P] digested pSB1C3-R21(co) in [S/P]
Friday 3				Transformation of July 2 ligation product pSB1C3-R21(co)-Rz21(co)

Week 8 : July 6 – July 10
Date	Group 1	Group 2	Group 3	Group 4
Monday 6				Colony PCR on July 3 R21(co)-Rz21(co) clones. No bands were observed
Tuesday 7			Extraction of June 30 plasmid pSB1C3-S21WT(ori)-R21(ori)-Rz21(ori) (21Lysis(o_o_o)) Restriction digestion with [X/P]	Redo July 6's colony PCR with other colonies. No bands were observed
Wednesday 8			Redo the restriction digestion of 21Lysis(o_o_o) with [X/P]
Thursday 9			Gel purification of July 8 [X/P] digested 21Lysis(o_o_o)
Friday 10			Ligation of the [X/P] digested 21Lysis(o_o_o) insert into [X/P] digested pSB1C3 vector Transformation	Restriction analysis on July 3 R21(co)-Rz21(co) clones. The analysis confirmed the presence of the R21(co)-Rz21(co) insert

Week 9 : July 13 – July 18
Date	Group 1	Group 2	Group 3	Group 4
Monday 13			Colony PCR on July 10 21Lysis(o_o_o) clones. No bands were observed
Tuesday 14			Restriction analysis on the 21Lysis(o_o_o) clones. The analysis confirmed the presence of the 21Lysis(o_o_o) insert	Extraction of the plasmid pSB1C3-R21(co)-Rz21(co) Restriction digestion with [X/P]
Wednesday 15				Gel purification of July 14 [X/P] digested R21(co)-Rz21(co)
Thursday 16
Friday 17
Saturday 18				Extraction of the plasmids pSB1C3-S21mut(ori) and pSB1C3-S21mut(co) Restriction digestion wth [S/P]

Week 10 : July 20 – July 25
Date	Group 1	Group 2	Group 3	Group 4
Monday 20				Gel purification of July 18 [S/P] digested pSB1C3-S21mut(ori) and pSB1C3-S21mut(co) Ligation of July 15 [X/P] digested R21(co)-Rz21(co) insert into [S/P] digested pSB1C3-S21mut(ori) Ligation of July 15 [X/P] digested R21(co)-Rz21(co) insert into [S/P] digested pSB1C3-S21mut(co)
Tuesday 21		Transformation of pGOv4-Ribo_S21WT(co), pGOv4-Ribo_R21(co) and pGOv4-Ribo_Rz21(co)		Transformation of July 20 ligation product pSB1C3-S21mut(ori)-R21(co)-Rz21(co) (21Lysis_Sm(o_c_c)) Transformation of July 20 ligation product pSB1C3-S21mut(co)-R21(co)-Rz21(co) (21Lysis_Sm(c_c_c))
Wednesday 22			Redo the transformation of pGOv4-Ribo_R21(co)	Colony PCR on the 21Lysis_Sm(o_c_c) clones revealed that the colonies contained inserts of the wrong sizes Colony PCR on the 21Lysis_Sm(c_c_c) clones showed bands of the expected size Redo the restriction digestion of R21(co)-Rz21(co) with [S/P] Gel purification
Thursday 23			Extraction of the plasmids pGOv4-Ribo_S21WT(co), pGOv4-Ribo_R21(co) and pGOv4-Ribo_Rz21(co) Restriction digestion of Ribo_S21WT(co) with [E/P] & [E/S], Ribo_R21(co) with [E/P] & [X/P] and Ribo_Rz21(co) with [E/P] & [X/P]	Redo the ligation of the [X/P] digested R21(co)-Rz21(co) insert into [S/P] digested pSB1C3-S21mut(ori) Transformation Ligation of [X/P] digested BBa_I13504 (GFP) insert into [S/P] digested 21Lysis(o_o_o)
Friday 24				Colony PCR on 21Lysis_Sm(o_c_c) clones showed bands of the expected size
Saturday 25

Week 11 : July 27 – August 1
Date	Group 1	Group 2	Group 3	Group 4
Monday 27
Tuesday 28			Extraction of the plasmid pGOv4-Ribo_Rz21(co) Redo the restriction digestion of Ribo_S21WT(co) and Ribo_R21(co) with [E/P]
Wednesday 29			Ligation of the [E/P] digested Ribo_S21WT(co) insert into the [E/P] digested pSB1C3 vector [E/P] & transformation Ligation of the [E/P] digested Ribo_R21(co) insert into [E/P] digested pSB1C3 Extraction of the plasmids pGOv4-Ribo_S21WT(co), pGOv4-Ribo_R21(co) and pGOv4-Ribo_Rz21(co) Restriction digestion of Ribo_S21WT(co) with [E/S], Ribo_R21(co) with [X/P] and Ribo_Rz21(co) with [E/P] Gel purification	Redo the ligation of the [X/P] digested GFP insert into the [S/P] digested pSB1C3-21Lysis(o_o_o) Transformation
Thursday 30			Restriction digestion of Ribo_R21(co) with [X/P]	Colony PCR on 21Lysis(o_o_o)-GFP clones showed bands of the expected size
Friday 31				Restriction digestion of 21Lysis_Sm(o_c_c) with [X/P] Gel purification Redo the extraction of the plasmid pSB1C3-21Lysis_Sm(c_c_c) Restriction digestion of 21Lysis_Sm(c_c_c) with [X/P] Gel purification
Saturday 1

Week 12 : August 3 – 8
Date	Group 1	Group 2	Group 3	Group 4
Monday 3				Extraction of plasmid pGOV4-S21WT(co)/li> Restriction digestion with [E/S] Gel purification Ligation of the [E/S] digested S21WT(co) and July 22 [X/P] digested R21(co)-Rz21(co) into July 22 [E/P] digested pSB1C3 Extraction of the plasmid pGOv4-lacIq (pLacIQ_LacI_L8-UV5 lac) Restriction digestion with [E/S] Gel purification Ligation of the [E/S] digested lacIq and [X/P] digested 21Lysis_Sm(o_c_c) into [E/P] digested pSB1C3 Ligation of the [E/S] digested lacIq promoter cassette and [X/P] digested 21Lysis_Sm(c_c_c) into [E/P] digested pSB1C3
Tuesday 4				Transformation of Aug 3 ligation product
Wednesday 5				Colony PCR on Aug 4 transformed pSB1C3-21Lysis(c_c_c). The clones contained inserts of the expected size Colony PCR on Aug 4 transformed pSB1C3-lacIq-21Lysis_Sm(o_c_c). The clones contained the wrong inserts Colony PCR on Aug 4 transformed pSB1C3-lacIq-21Lysis_Sm(c_c_c). The clones contained the wrong inserts
Thursday 6				Restriction digestion of 21Lysis(c_c_c) with [E/X] Gel purification Ligation of the [E/S] digested lacIq into the [E/X] digested pSB1C3-21Lysis(c_c_c) Restriction digestion of 21Lysis_Sm(o_c_c) and 21Lysis_Sm(c_c_c) with [E/X] Gel purification Redo the ligation of the [E/S] digested lacIq into the [E/X] digested pSB1C3-21Lysis_Sm(o_c_c) and pSB1C3-21Lysis_Sm(c_c_c) respectively
Friday 7				Transformation of Aug 6 ligation product Redo the restriction digestion with [E/S] on the plasmid pGOv4-S21WT(co) Gel purification
Saturday 8				* Colony PCR on Aug 7 transformed cells. The clones lacIq-21Lysis(c_c_c), lacIq-21Lysis_Sm(o_c_c) and lacIq-21Lysis_Sm(c_c_c) all contained inserts of the expected sizes Ligation of the [E/S] digested S21WT(co) into the [E/S] digested pSB1C3 Transformation

Week 13 : August 10 – 16
Date	Group 1	Group 2	Group 3	Group 4
Monday 10				Colony PCR on Aug 8 transformed cells. The S21WT(co) clones contained inset of the expected size

ABOUT US

We are a diverse team of CityU undergraduates, working hard to create a better world.

LOCATION

Department of Biology and Chemistry,
City University of Hong Kong
Tat Chee Avenue, Kowloon,
Hong Kong SAR

CONTACT US

Email: cityu.igem2015@gmail.com
Tel: +852 34427654

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-				>
-				Safety
-			</a>
-		</li>
-		<li id="active"
-			class="wsite-menu-item-wrap"
-			>
-			<a href="notebook.html"
-				class="wsite-menu-item"
-				>
-				Notebook
-			</a>
-		</li>
-</ul>
-</div></td>
-					</tr>
-				</table>
-			</div><!-- end container -->
-		</div><!-- end nav-wrap -->
 		<div id="main-wrap">
 			<div class="container">
 				<div id='wsite-content' class='wsite-elements wsite-not-footer'>
-<center><a>
-<img src="https://static.igem.org/mediawiki/2015/8/8c/CityU_2015_Group_photo.png" alt="Picture" style="" />
-</a></center>
 <div class="wsite-spacer" style="height:50px;"></div>
@@ Line 879: / Line 559: @@
 		</div><!-- end main-wrap -->
-    <div id="footer-wrap">
-        <div class="container">
-       		Create a <a target="_top" href="http://www.weebly.com/">free web site</a> with <a target="_top" href="http://www.weebly.com/" title="site web gratuit">Weebly</a>
-        </div><!-- end container -->
-    </div><!-- end footer-wrap -->
 	</body>
 </html>
-{{:Team:CityU_HK/Template/CSS}}
+{{:Team:CityU_HK/Template/css}}
+{{:Team:CityU_HK/Template/Footer}}

Difference between revisions of "Team:CityU HK/Notebook/Lysis plasmid (phage 21)"

Revision as of 07:57, 10 September 2015

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