Team:CityU HK/Notebook

City University of Hong Kong 2015

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Notebook

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lacZY plasmid

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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

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Lysis plasmid (λ phage)

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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		Extraction of the plasmid pGOv4-Rzλ(co)	Extraction of the plasmid pGOv4-Rλ(co)
Thursday 11	Preparation of the plasmid pSB1C3 Restriction digestion with [E/P]	Restriction digestion with [E/P] on the plasmid pGOv4-Rzλ(co) Gel purification	Restriction digestion with [E/P] on the plasmid pGOv4-Rλ(co) Gel purification
Friday 12		Ligation of the [E/P] digested Rzλ(co) insert (from June 11) with the [E/P] digested vector pSB1C3 Transformation of the ligaiton product into competent E. coli cells

Week 5 : June 15 – 19
Date	Group 1	Group 2	Group 3	Group 4
Monday 15
Tuesday 16		Colony PCR on June 12 transformed cells (Result: no bands amplified)	Ligation of the [E/P] digested Rλ(co) insert (from June 12) into the [E/P] digested vector pSB1C3 Transformation of the ligation product into competent E. coli cells
Wednesday 17			Colony PCR on June 16 transformed cells (Result: no bands amplified)
Thursday 18		Redo the ligation of the [E/P] digested Rzλ(co) insert into the [E/P] digested vector pSB1C3 Transformation of the ligation product into competent E. coli cells	Redo the extraction of the plasmid pGOv4-Rλ(co) Restriction digestion with [E/P]
Friday 19		Colony PCR on June 18 transformed cells (Result: seen bands of the expected size)	Gel purification of June 18 digest

Week 6 : June 22 – 26
Date	Group 1	Group 2	Group 3	Group 4
Monday 22
Tuesday 23			Redo the extraction of the plasmid pGOv4-Rλ(co) Restriction digestion with [E/P] Gel purification
Wednesday 24
Thursday 25			Ligation of the [E/P] digested Rλ(co) insert into the [E/P] digested pSB1C3 vector Transformation of the ligation product into competent E. coli cells
Friday 26			Colony PCR on June 25 transformed cells (Result: showed no bands)

Week 7 : June 29 – July 3
Date	Group 1	Group 2	Group 3	Group 4
Monday 29		Extraction of the plasmid pSB1C3-Rzλ(co) (from June 18 transformed cells)	Colony PCR on June 25 transformed cells (Result: again showed no bands)
Tuesday 30			Sequencing result confirmed the presence of Rλ(co) insert in pSB1C3
Wednesday 1
Thursday 2	PCR amplification of the BBa_K124017 SλWT(ori) gene	PCR amplification of the BBa_K124017 Rλ(ori) gene Rλ(ori) PCR product purification Transformation of pGOv4-SλWT(co) into competent E. coli cells	PCR amplification of the BBa_K124017 Rzλ(ori) gene Rzλ(ori) PCR product purification Restriction digestion with [E/P]	PCR amplification of the BBa_K124017 Rλ(ori) gene Rλ(ori) PCR product purification
Friday 3	Restriction digestion of pSB1C3 vector with [E/X]	Restriction digestion of Rλ(co) with [E/P] Gel purification	[1] Gel purification of July 2 [E/P] digested Rzλ(ori) Ligation of the [E/P] digested Rzλ(ori) insert into [E/P] digested pSB1C3 vector [2] Extraction of the plasmid pSB1C3-Rλ(co) (from June 25 transformed cells) Restriction digestion with [S/P] Gel purification [3] Restriction digestion of June 2 Rzλ(ori) with [X/P] & Heat kill Ligation of [X/P] digested Rzλ(ori) insert into [S/P] digested pSB1C3-Rλ(co) vector	Extraction of the plasmid pSB1C3-Rλ(co) (from Gp3 June 25 transformed cells)

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