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Lab Log

lacZY plasmid

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	==introduction==
Tuesday 19
Wednesday 20	PCR amplification of the BBa_S04055 lacZ gene	PCR amplification of the BBa_S04055 lacZ gene	PCR amplification of the BBa_S04055 lacY gene	PCR amplification of the BBa_S04055 lacY gene
Thursday 21	Gel electrophoresis of the amplicon lacZ PCR product purification Restriction digestion with [E/P] of the vector pSB1C3 & lacZ	Gel electrophoresis of the amplicon lacZ PCR product purification Restriction digestion with [E/P] of the vector pSB1C3 & lacZ	Gel electrophoresis of the amplicon lacY PCR product purification Restriction digestion with [E/P] of the vector pSB1C3 & lacY	Gel electrophoresis of the amplicon lacY PCR product purification Restriction digestion with [E/P] of the vector pSB1C3 & lacY
Friday 22	Gel purification of vector pSB1C3 [E/P] digest Ligation of the [E/P] digested lacZ insert into the [E/P] digested vector pSB1C3	Gel purification of vector pSB1C3 [E/P] digest Ligation of the [E/P] digested lacZ insert into the [E/P] digested vector pSB1C3	Gel purification of vector pSB1C3 [E/P] digest Ligation of the [E/P] digested lacY insert into the [E/P] digested vector pSB1C3	Gel purification of vector pSB1C3 [E/P] digest Ligation of the [E/P] digested lacY insert into the [E/P] digested vector pSB1C3

Week 2 : May 25 – 29
Date	Group 1	Group 2	Group 3	Group 4
Monday 25
Tuesday 26	Transformation of May 22 ligation product into competent E. coli cells	Transformation of May 22 ligation product into competent E. coli cells	Transformation of May 22 ligation product into competent E. coli cells	Transformation of May 22 ligation product into competent E. coli cells
Wednesday 27	Colony PCR of May 26 transformed cells	Colony PCR of May 26 transformed cells	Colony PCR of May 26 transformed cells	Colony PCR of May 26 transformed cells
Thursday 28	Extraction of the plasmid pSB1C3-BBa_J23100 Restriction digestion with [S/P] of the vector pSB1C3-BBa_J23100	Extraction of the plasmid pSB1C3-BBa_J23100 Restriction digestion with [S/P] of the vector pSB1C3-BBa_J23100	Extraction of the plasmid pSB1C3-BBa_J23100 Restriction digestion with [S/P] of the vector pSB1C3-BBa_J23100	Extraction of the plasmid pSB1C3-BBa_J23100 Restriction digestion with [S/P] of the vector pSB1C3-BBa_J23100
Friday 29	Gel purification of May 28 digest	Gel purification of May 28 digest	Gel purification of May 28 digest	Gel purification of May 28 digest

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	Extraction of the plasmid pSB1C3-lacZ (from May 26 transformed cells) Restriction digestion with [X/P] of the insert lacZ Gel purification	Extraction of the plasmid pSB1C3-lacY (from May 26 transformed cells) Restriction digestion with [X/P] of the insert lacY Gel purification	Extraction of the plasmid pSB1C3-lacZ from Gp1 Restriction digestion with [E/S] of the insert lacZ Gel purification	Extraction of the plasmid pSB1C3-lacY from Gp1 Restriction digestion with [E/S] of the insert lacY Gel purification
Tuesday 2	Redo the work on June 1	Redo the work on June 1	Redo the work on June 1	Redo the work on June 1
Wednesday 3	Ligation of Gp1 [X/P] digested lacZ insert (from June 2) into [S/P] digested vector pSB1C3-BBa_J23100 (from May 29) Transformation		Ligation of Gp2 [E/S] digested lacZ insert (from June 2) into Gp4 [E/X] digested vector pSB1C3-lacY(from June 2) Transformation
Thursday 4	Colony PCR on June 3 transformed cells		Colony PCR on June 3 transformed cells
Friday 5	Extraction of plasmid pSB1C3-BBa_J23100-lacZ (from June 3 transformed cells) Restriction digestion with [S/P] of the vector pSB1C3-BBa_J23100-lacZ Gel purification		Extraction of plasmid pSB1C3-lacZ-lacY (from June 3 transformed cells) Restriction digestion with [X/P] of the insert lacZ-lacY Gel purification

Week 4 : June 8 – 12
Date	Group 1	Group 3	Group 2	Group 4
Monday 8	Ligation of Gp3 [X/P] digested lacY insert (from June 2) into [S/P] digested vector pSB1C3-BBa_J23100-lacZ (from June 5) Transformation		Ligation of [X/P] digested lacZ-lacY insert (from June 5) into [S/P] digested vector pSB1C3-BBa_J23100 (from May 29) Transformation
Tuesday 9	Colony PCR of June 8 transformed cells (failed)		Colony PCR of June 8 transformed cells (failed)
Wednesday 10	Extraction of the plasmid pSB1C3-BBa_J23100-lacZ-lacY (from June 8 transformed cells) Restriction analysis with [E/P] for confirmation of the insert size		Extraction of the plasmid pSB1C3-BBa_J23100-lacZ-lacY (from June 8 transformed cells) Restriction analysis with [E/P] for confirmation of the insert size

Characterization of the lacZY plasmid

Week 8 : July 6 – 10
Date		Remarks
Monday 6
Tuesday 7	RNA extraction from the E. coli cells harboring the recombinant plasmid pSB1C3-BBa_J23100- lacZ-lacY	Not successful
Wednesday 8	Redo RNA extraction	Not successful
Thursday 9
Friday 10	Redo RNA extraction	Not successful

Week 9 : July 13 – 18
Date		Remarks
Monday 13	Redo the RNA extraction	Successful
Tuesday 14	Extract RNA from control E. coli cells	Successful
Wednesday 15	Redo RNA extraction from control E. coli cells Perform RT-PCR on purified RNA from recombinant and control E. coli	Successful
Thursday 16	Check the size of the RT-PCR product using gel electrophoresis	Successful
Friday 17	Prepare Z-buffer
Saturday 18	Perform qPCR on the control cDNA	Successful

Week 10 : July 20 – 25
Date		Remarks
Monday 20	Perform q-PCR on recombinant cDNA	Successful
Tuesday 21	Perform ONPG assay -characterization on the expression level of LacZ protein
Wednesday 22
Thursday 23
Friday 24

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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