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

Notebook - City University of Hong Kong 2015

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Notebook
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
    		•
    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)
    		•