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Revision as of 18:20, 16 September 2015

Description - iGEM2015 wiki /* ****** Back to top button ******* */

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
    		•
    Interlab study

    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
    		•