Team:CityU HK/Notebook

Notebook - City University of Hong Kong 2015

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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
    Lysis plasmid (λ phage)

    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
    		•