Team:CityU HK/Notebook/Lysis plasmid (phage 21)

Notebook
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
    		•