Team:CityU HK/Notebook

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Notebook

• LacZY plasmid
• Lysis plasmid (λ phage)
• Lysis plasmid (Phage 21)
• Interlab study

Lab Log

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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	Redo 11/6’s work

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)

Week 8 : July 6 – July 10
Date	Group 1	Group 2	Group 3	Group 4
Monday 6	Gel purification of the [X/P] digested pSB1C3 vector isolated on July 3 PCR amplification of the BBa_K124017 Rzλ(ori) gene using a F-primer with SacII restriction site		Transformation of the ligation product pSB1C3-Rλ(co)-Rzλ(ori) (from July 3 [3])	Restriction digestion of July 3 Rλ(co) with [S/P] Gel purification Restriction digestion of Rzλ(co) (from Gp2 June 29) with [X/P] Gel purification
Tuesday 7	Gel electrophoresis of the SacII digested Rzλ(ori) PCR amplicon PCR amplification of SλWT(ori) using a new primer pair	Extraction of the plasmid pGOv4-SλWT(co) Restriction digestion with [X] on July 3 [P] digested Rλ(ori)	Colony PCR on July 6 Rλ(co)-Rzλ(ori) clones showed no bands Redo July 3’s ligation of [X/P] digested Rzλ(ori) insert into [S/P] digested pSB1C3-Rλ(co) vector Transformation	Ligation of the [X/P] digested Rzλ(co) insert into the [S/P] digested pSB1C3-Rλ(co) vector Transformation
Wednesday 8	PCR purification of July 6 SacII Rzλ(ori) PCR product	Ligation of [X/P] digested Rλ(ori) into Gp1 July 6 [X/P] Transformation of 7/7’s ligation product Rλ(ori) in pSB1C3	Colony PCR on July 7 Rλ(co)-Rzλ(ori) clones still showed no bands	Colony PCR on July 7 Rλ(co)-Rzλ(co) clones showed no bands
Thursday 9	Restriction digestion of SacII Rzλ(ori) with [SacII/P] Restriction digestion of Gp2 July 7 SλWT(co) with [E/S] Gel electrophoresis of July 7 SλWT(ori) PCR purification of SλWT(ori)	Colony PCR on Rλ(ori) (from July 8 transformed cells) Restriction digestion of July 7 SλWT(co) with [E/P]	Ligation of [X/P] July 3 digested Rzλ(ori) insert into [X/P] digested pSB1C3 vector Transformation
Friday 10	Restriction digestion of SλWT(ori) with [X/P] & Gel purification Ligation of [X/P] digested SλWT(ori) insert into July 6 [X/P] digested pSB1C3 vector Transformation Gel purification of July 9 [E/S] digested SλWT(co)	Gel purification of July 9 [E/P] digested SλWT(co)	Colony PCR on July 9 Rzλ(ori) clones showed no bands Restriction analysis on July 7 transformed cells Another redo of the ligation of July 3 [X/P] digested Rzλ(ori) insert into July 3 [S/P] digested pSB1C3-Rλ(co) Transformation	Extraction of the plasmid pSB1C3-Rλ(co)-Rzλ(co) (from July 7 transformed cells) Restriction analysis of Rλ(co)-Rzλ(co) showed bands of the expected size

Week 9 : July 13 – July 18
Date	Group 1	Group 2	Group 3	Group 4
Monday 13	Colony PCR on SλWT(ori) clones showed bands of the expected size Ligation of [E/S] digested SλWT(co) insert into pSB1C3 vector Transformation	Extraction of the plasmid pSB1C3-Rλ(ori) Restriction digestion with [X/P] Gel purification	Colony PCR on 10/7 transformed cells pSB1C3-Rλ(co)-Rzλ(ori)
Tuesday 14	Colony PCR on SλWT(co) clones showed bands of the expected size Restriction digestion of Gp2 Rλ(ori) with [X/SacII]	Restriction analysis of [X/P] digested Rλ(ori) Restriction digestion of Rzλ(co) with [X/P] Gel purification		Extraction of the plasmid pSB1C3-Rλ(co)-Rzλ(co) Restriction digestion with [X/P]
Wednesday 15	Gel purification of July 14 [X/SacII] digested Rλ(ori) Ligation between [E/S] digested SλWT(co), [X/SacII] digested Rλ(ori), [SacII/P] digested Rzλ(ori) and [E/P] digested pSB1C3 backbone Transformation	Extraction of the plasmid pSB1C3-Rλ(ori) Restriction digestion with [S/P]	Extraction of the plasmid pSB1C3-Rλ(co) Restriction digestion of Rλ(co) with [E/S] Restriction digestion of [E/X] digested Rzλ(ori) with CIP & Gel purification Ligation of the [E/S] digested Rλ(co) insert into [E/X] digested pSB1C3-Rzλ(ori) Transformation	Gel purification of July 14 [X/P] digested Rλ(co)-Rzλ(co)
Thursday 16	Colony PCR on SλWT(co)- Rλ(ori)-Rzλ(ori) showed bands believed to be the desired insert	Gel purification of July 15 [S/P] digested Rλ(ori) Ligation of the [X/P] digested Rzλ(co) insert into [S/P] digestd pSB1C3-Rλ(ori) vector	Colony PCR on July 15 transformed cells Redo the ligation of [E/S] digested Rλ(co) insert into [E/X] digested pSB1C3-Rzλ(ori) Transformation
Friday 17		Transformation of July 16 ligation product pSB1C3-Rλ(ori)-Rzλ(co)	Colony PCR on 16/7 transformed cells Restriction digestion of Sλ(co) with [E/S] & Gel purification Redo the ligation of [E/S] digested Rλ(co) insert into [E/X] digested pSB1C3-Rzλ(ori) Transformation
Saturday 18				Extraction of the plasmids pSB1C3-Sλmut(ori) and pSB1C3-Sλmut(co) Restriction digestion with [S/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.

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-Sλmut(ori) and pSB1C3-Sλmut(co) Ligation of July 15 [X/P] digested Rλ(co)-Rzλ(co) insert into the [S/P] digested pSB1C3-Sλmut(ori) vector Ligation of July 15 [X/P] digested Rλ(co)-Rzλ(co) insert into the [S/P] digested pSB1C3-Sλmut(co) vector
Tuesday 21		Transformation of pGOv4-Ribo_Sλ(co), pGOv4-Ribo_Rλ(co) and pGOv4-Ribo_Rzλ(co) into competent E. coli cells Transformation of pGOv4-SλWT(co)-Rλ(co)-Rzλ(co) (λLysis(c_c_c))		Transformation of the pSB1C3-Sλmut(ori)-Rλ(co)-Rzλ(co) (λLysis_Sm(o_c_c)) Transformation of the pSB1C3-Sλmut(co)-Rλ(co)-Rzλ(co) (λLysis_Sm(c_c_c))
Wednesday 22	Genomic DNA extraction of BL21(DE3) E. coli cells PCR amplification of the Sλ(ori), Rλ(ori) and Rzλ(ori) genes using the extracted genomic DNA as template		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 E. coli cells	Colony PCR on λLysis_Sm(o_c_c) clones showed bands of the expected size Colony PCR on c clones revealed that that the colonies contained inserts of the wrong sizes Redo the restriction digestion of Rλ(co)-Rzλ(co) with [X/P] & Gel purification
Thursday 23	Extraction of the plasmids pGOv4-Ribo_Sλ(co), pGOv4-Ribo_Rλ(co) and pGOv4-Ribo_Rzλ(co) Restriction digestion of the extracted plasmids with [E/P] Gel purification		Extraction of the plasmid pGOv4-λLysis(c_c_c) Restriction digestion with [E/P]	Redo the ligation of the [X/P] digested Rλ(co)-Rzλ(co) insert into the [S/P] digested pSB1C3-Sλmut(co) vector Transformation
Friday 24	Ligation of the [E/P] digested Ribo_Sλ(co) and Ribo_Sλ(co) inserts into [E/P] digested pSB1C3 respectively Transformation Restriction digestion of the Sλ(ori), Rλ(ori) and Rzλ(ori) amplicons with [X/P]			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
Saturday 25	Colony PCR on July 24 transformed Ribo_Sλ and Ribo_Rλ clones Ligation of the [X/P] digested Sλ(ori), Rλ(ori) and Rzλ(ori) inserts into [X/P] digested pSB1C3 respectively Transformation

Week 11 : July 27 – August 1
Date	Group 1	Group 2	Group 3	Group 4
Monday 27
Tuesday 28	Colony PCR on Sλ(ori), Rλ(ori) and Rzλ(ori) clones showed bands of the expected size		Restriction digestion of Ribo_Rλ(co) with [E/P]
Wednesday 29				Restriction digestion of λLysis_Sm(o_c_c) with [X/P] Gel purification
Thursday 30
Friday 31			Redo the restriction digestion of Ribo_Rλ(co) with [E/P]	Redo the extraction of the plasmid pSB1C3-λLysis_Sm(o_c_c) Redo the restriction digestion with [X/P] Gel purification
Saturday 1			Extraction of the plasmid pSB1C3-BBa_J23100 Restriction digestion with [E/S] and [E/P] respectively & Gel purification Restriction digestion of λLysis(c_c_c) with [X/P] & Gel purification

Week 12 : August 3 – August 9
Date	Group 1	Group 2	Group 3	Group 4
Monday 3				Redo the ligation of the [X/P] digested Rλ(co)-Rzλ(co) insert into the [S/P] digested pSB1C3-Sλmut(co) vector 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 into [E/S] digested pSB1C3 Ligation of the [E/S] digested lacIq and [X/P] digested λLysis_Sm(o_c_c) into [E/P] digested pSB1C3
Tuesday 4				Transformation of Aug 3 ligation product
Wednesday 5				The Aug 4 transformed pSB1C3-lacIq showed no growth Colony PCR on Aug 4 transformed λLysis_Sm(c_c_c) clones. The clones contained inserts of the correct size Colony PCR on Aug 4 transformed lacIq-λLysis_Sm(o_c_c) clones. The clones contained inserts of the wrong sizes
Thursday 6				Restriction digestion of λLysis_Sm(o_c_c) with [E/X] Gel purification Redo the ligation of [E/S] digested lacIq into the [E/X] digested pSB1C3-λLysis_Sm(c_c_c) Redo the ligation of the [E/S] digested lacIq into [E/S] digested pSB1C3
Friday 7				Redo the ligation of the [E/S] digested lacIq into the [E/X] digested pSB1C3-λLysis_Sm(o_c_c) Transformation
Saturday 8				Restriction digestion of λLysis_Sm(c_c_c) with [E/X] Gel purification
Sunday 9				Colony PCR on Aug 7 transformed cells. The lacIq clones contained inserts of the correct size. The lacIq-λLysis_Sm(o_c_c) clones showed no bands Redo the restriction digestion of λLysis_Sm(c_c_c) with [E/X] Gel purification Ligation of the [E/S] digested lacIq into the [E/X] digested pSB1C3-λLysis_Sm(c_c_c)

Week 13 : August 10 – August 16
Date	Group 1	Group 2	Group 3	Group 4
Monday 10				Redo the colony PCR on Aug 7 transformed lacIq-λLysis_Sm(o_c_c) cells. The clones contained inserts of the wrong sizes Transformation of Aug 9 ligation product pSB1C3-lacIq-λLysis_Sm(c_c_c)

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Lysis plasmid (phage 21)

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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

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Interlab study

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Keys to the table:

-[?/?]: Names of restriction enzyme used for digestion:; E, EcoRI; X, XbaI; S, SpeI; P, PstI

-The host cells used for transformation were competent JM109 E. coli cells

Week 8 : July 6 – 10
Monday 6	Transformation of pSB1C3-BBa_J23101, pSB1C3-BBa_J23106 and pSB1C3-BBa_J23117, and pSB1A2-BBa_I13504 into competent E. coli cells
Tuesday 7
Wednesday 8	Extraction of the plasmids pSB1C3-BBa_J23101, pSB1C3-BBa_J23106, pSB1C3-BBa_J23117 and pSB1A2-BBa_I13504 by mini-prep
Thursday 9
Friday 10

Week 9 : July 13 – 18
Monday 13	Transformation of plasmid pSB1C3-BBa_I13504 into competent E. coli cells
Tuesday 14	Restriction digestion with [S/P] of the plasmid pSB1C3-BBa_J23101 Restriction digestion with [S/P] of the plasmid pSB1C3-BBa_J23106 Restriction digestion with [S/P] of the plasmid pSB1C3-BBa_J23117 Redo the transformation of plasmids pSB1C3-BBa_I20270 and pSB1C3-BBa_R0040
Wednesday 15	Extraction of plasmid pSB1C3-BBa_I13504 Restriction digestion with [X/P] of the plasmid pSB1C3-BBa_I13504
Thursday 16	Extraction of plasmids pSB1C3-BBa_I20270 and pSB1C3-BBa_R0040 Gel purification of the [X/P] digested I13504 Gel purification of July 14 [S/P] digested vectors pSB1C3-BBa_J23101, pSB1C3-BBa_J23106 and pSB1C3-BBa_J23117
Friday 17	Ligation of the [X/P] digested BBa_I13504 insert into [S/P] digested vector pSB1C3-BBa_J23101 Ligation of the [X/P] digested BBa_I13504 insert into [S/P] digested vector pSB1C3-BBa_J23106 Ligation of the [X/P] digested BBa_I13504 insert into [S/P] digested vector pSB1C3-BBa_J23117 Transformation of the 3 ligation product
Saturday 18

Week 10 : July 20 – 25
Monday 20	Redo the transformation of all July 17 ligation product
Tuesday 21	Redo the transformation of July 17 pSB1C3-BBa_J23101-I13504 and pSB1C3-BBa_J23117-BBa_I13504 ligation product
Wednesday 22	Extraction of plasmid pSB1C3-BBa_J23106-I13504 Restriction analysis with [E/P] of the plasmid pSB1C3-BBa_J23106-BBa_I13504
Thursday 23	Restriction analysis with [E/P] of the plasmids pSB1C3-BBa_J23101-I13504 and pSB1C3-BBa_J23117-I13504
Friday 24
Saturday 25

Week 11 : July 27 – August 1
Monday 27
Tuesday 28	Checking of O.D A600, A511 - Positive control (I20270), negative control (R0040) and the J23101, J23106 and J23117 Redo the transformation of pSB1C3-BBa_J23117
Wednesday 29
Thursday 30	Extraction of the plasmid pSB1C3-BBa_J23117 Restriction digestion [S/P] of pSB1C3-BBa_J23117 Restriction digestion [X/P] of pSB1A2-BBa_I13504 Gel purification
Friday 31	Ligation of the [X/P] digested BBa_I13504 insert into the [S/P] digested pSB1C3-J23117 Transformation
Saturday 1	Extraction of the plasmids pSB1C3-BBa_J23117 and pSB1A2_BBa_I13504 Extraction of the plasmids pSB1C3-BBa_J23117 and pSB1A2_BBa_I13504

Week 12 : August 3 – 8
Monday 3	Redo the extraction of the plasmids pSB1C3-BBa_J23117 and pSB1A2_BBa_I13504 Restriction analysis [E/P]
Tuesday 4	Redo the ligation of the [X/P] digested BBa_I13504 into the [S/P] digested pSB1C3-BBa_J23117

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