iGEM 2015 — Team CityU_HK

# Team:CityU HK/Notebook

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* NOTEBOOK

  + [LAB LOG](https://2015.igem.org/Team%3ACityU_HK/Notebook)
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* OUTREACH

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* [INTERLAB](//2015.igem.org/Team%3ACityU_HK/Interlab)

Description - iGEM2015 wiki

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[ ]

## Notebook

* [LacZY plasmid](#laczy)
* [Lysis plasmid (λ phage)](#lysis-lambda)
* [Lysis plasmid (Phage 21)](#lysis-21)
* [Interlab study](#interlab)

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 (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 | - Redo 11/6’s work |  |

| 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) |

---

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 |

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