Difference between revisions of "Team:CityU HK/Notebook/lacZY plasmid"

Latest revision as of 07:54, 10 September 2015

Notebook

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

ABOUT US

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LOCATION

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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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Difference between revisions of "Team:CityU HK/Notebook/lacZY plasmid"

Latest revision as of 07:54, 10 September 2015

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