iGEM 2015 — Team Hong_Kong_HKU

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# Team:Hong Kong HKU/Notebook

Http://2014hs.igem.org/Team:UCL Academy - 2014.igem.org

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| ![](http://2015.igem.org/wiki/images/f/f0/Notebook_banner_hku.jpeg) |

### NOTEBOOK

|  |  |  |  |
| --- | --- | --- | --- |
| [![](http://2015.igem.org/wiki/images/d/de/W1_hku.jpeg)](#week1) | [![](http://2015.igem.org/wiki/images/b/be/W2_hku.jpeg)](#week2) | [![](http://2015.igem.org/wiki/images/2/2e/W3_hku.jpeg)](#week3) | [![](http://2015.igem.org/wiki/images/6/64/W4_hku.jpeg)](#week4) |
| [![](http://2015.igem.org/wiki/images/a/a8/W5_hku.jpeg)](#week5) | [![](http://2015.igem.org/wiki/images/e/e2/W6_hku.jpeg)](#week6) | [![](http://2015.igem.org/wiki/images/3/30/W7_hku.jpeg)](#week7) | [![](http://2015.igem.org/wiki/images/3/37/W8_hku.jpeg)](#week8) |
| [![](http://2015.igem.org/wiki/images/4/45/W9_hku.jpeg)](#week9) | [![](http://2015.igem.org/wiki/images/d/de/W10_hku.jpeg)](#week10) | [![](http://2015.igem.org/wiki/images/c/cf/W11_hku.jpeg)](#week11) | [![](http://2015.igem.org/wiki/images/4/49/W12_hku.jpeg)](#week12) |
| [![](http://2015.igem.org/wiki/images/b/b1/W13_hku.jpeg)](#week13) |  |  |  |

#### Week 1

## 22 JUN 2015

1. 200 mL of LB broth was prepared.

## 23 JUN 2015

1. 100 mL of 0.1M CaCl2 and 100 mL of 0.1M CaCl2/15% glycerol were prepared.

2. 5 mL of MG1655 cell culture was inoculated overnight.

## 24 JUN 2015

1. MG1655 competent cells were made.

2. LB agar plates were prepared, half of them with CM30 and half of them with no antibiotics.

## 25 JUN 2015

1. 12 LB agar plates with CM30 were prepared.

2. Competent cells prepared on 24/6 were tested by using the Competent Cell Test Kit with CM30 plates.

## 26 JUN 2015

1. The competency test result was confirmed that no colonies could be found on any plates.

2. Competent cells prepared on 24/6 were tested again by using the Competent Cell Test Kit but with AmpR plates with pGm-wi.

3. The following BioBricks were transformed using DH5β competent cells borrowed,

- BBa\_K588001: >30 colonies

- BBa\_K1218011: >10 colonies

- BBa\_C0080: >30 colonies

- BBa\_J23106: no colonies

- BBa\_J61100: no colonies.

[##### Top](#Top)

#### Week 2

## 29 JUN 2015

1. The competency test result was confirmed that no colonies could be found on the AmpR plates with pGM-WT.

2. Transformation result was confirmed that

- BBa\_C0051, BBa\_K105004 and BBa\_B0015 plates showed >30 colonies

- BBa\_B0030 showed 2 colonies

- BBa\_J61100 and BBa\_R0051 showed no colonies.

3. The following BioBricks were transformed again using DH5β competent cells borrowed with CM30 plates,

- BBa\_B0030

- BBa\_J61100

- BBa\_R0051

4. Colonies of successfully transformed BioBricks were picked and incubated overnight.

5. 5 mL of MG1655 cell culture was inoculated overnight

## 30 JUN 2015

1. Transformation result was confirmed that

- BBa\_B0030 showed 2-3 colonies

- BBa\_R0051 showed >30 colonies

- BBa\_J61100 showed none

2. New MG1655 competent cells were prepared.

3. The following successfully transformed BioBricks underwent MiniPrep,

- BBa\_K105004

- BBa\_B0030

- BBa\_B0015

- BBa\_C0051

## 01 JUL 2015

1. The DNA concentration of the following BioBricks was measured by Nanodrop 1000

|  |  |
| --- | --- |
| BBa\_K105004 (i) | 223.9 ng/uL |
| BBa\_K105004 (ii) | 99.5 ng/uL |
| BBa\_K105004 (iii) | 97.2 ng/uL |
| BBa\_B0030 (i) | 64.7 ng/uL |
| BBa\_B0030 (ii) | 21.2 ng/uL |
| BBa\_B0015 (i) | 69.1 ng/uL |
| BBa\_B0015 (ii) | 91.1 ng/uL |
| BBa\_B0015 (iii) | 51.0 ng/uL |
| BBa\_C0051 (i) | 305.7 ng/uL |
| BBa\_C0051 (ii) | 232.9 ng/uL |
| BBa\_C0051 (iii) | 445.6 ng/uL |

2. The following BioBricks were transformed using DH5β competent cells borrowed with CM30 plates,

- BBa\_J23106: pC family member

- BBa\_C0080: araC (+ LVA)

- BBa\_K588001: ptrp

- BBa\_K1218011: cas 9

- BBa\_J61100

3. BBa\_K105004 was used to test MG1655 competent cells.

## 02 JUL 2015

1. Transformation result was confirm that

- BBa\_K588001: >30 colonies

- BBa\_K1218011: >10 colonies

- BBa\_C0080: >30 colonies

- BBa\_J23106: no colonies

- BBa\_J61100: no colonies

2. Competency test result was confirmed that no colonies could be found on the plate using BBa\_K105004.

3. BBa\_K577881 was resuspended from the kit and stored in a labelled Eppendorf tube.

## 03 JUL 2015

1. Requests were made to Headquarter for the following BioBricks

- BBa\_J61100

- BBa\_J119124 (trpR)

- BBa\_K512001 (CasABCDE12)

- BBa\_K786031 (T7 promoter - RSR - T7 terminator)

- BBa\_K786032 (Cas3 in E. coli Crispr system)

[##### Top](#Top)

#### Week 3

## 06 JUL 2015

1. The following BioBricks were transformed using DH5β competent cells borrowed onto CM30 plates,

- BBa\_J61100

- BBa\_J23106

- BBA\_K577881

2. MG1655 competent cells were tested by transforming pGM-WT on AmpR plates with DH10B competent cells.

3. Colonies of the successfully transformed BioBricks were picked from the plates and incubated overnight,

- BBa\_C0080

- BBa\_R0051

- BBa\_K588001

- BBa\_K1218011

- BBa\_B0030

4. New stock of MG1655 was prepared by streaking a new plate.

## 07 JUL 2015

1. Transformation result was confirmed that

- BBa\_J61100: no colonies

- BBa\_J23106: no colonies

- BBa\_K577881: >30 colonies

2. Competency test result was confirmed that

- our own MG1655 competent cells failed but others’ DH10B worked

3. The following BioBricks were miniprepped,

- BBa\_C0080

- BBa\_R0051

- BBa\_K588001

- BBa\_K1218011

- BBa\_B0030

4. New stock of MG1655 was prepared by streaking a new plate.

## 08 JUL 2015

1. 3 colonies of BBa\_K577881 were picked from the plate and incubated overnight.

## 09 JUL 2015

1. BBa\_K577881 was miniprepped.

2. DNA concentration of the following BioBricks was measured by Nanodrop 1000

|  |  |
| --- | --- |
| BBa\_C0080 (i) | 108.8 ng/uL |
| BBa\_C0080 (ii) | 165.0 ng/uL |
| BBa\_C0080 (iii) | 133.6 ng/uL |
| BBa\_R0051 (i) | 30.5 ng/uL, 32.6 ng/uL |
| BBa\_R0051 (ii) | 337.0 ng/uL, 333.3 ng/uL |
| BBa\_R0051 (iii) | 263.8 ng/uL, 258.4 ng/uL |
| BBa\_K588001 (i) | 187.3 ng/uL, 175.8 ng/uL |
| BBa\_K588001 (ii) | 18.6 ng/uL |
| BBa\_K588001 (iii) | 184.6 ng/uL, 182.8 ng/uL |
| BBa\_B0030 (i) | 1.6 ng/uL, 1.9 ng/uL |
| BBa\_B0030 (ii) | 12.1 ng/uL |
| BBa\_B0030 (iii) | 18.8 ng/uL |
| BBa\_K1218011 (i) | 28.7 ng/uL |
| BBa\_K1218011 (ii) | 280.7 ng/uL, 270.8 ng/uL |
| BBa\_K1218011 (iii) | 318.1 ng/uL |
| BBa\_K577881 (i) | 46.2 ng/uL |
| BBa\_K577881 (ii) | 23.5 ng/uL, 23.5 ng/uL |
| BBa\_K577881 (iii) | 53.7 ng/uL |

## 10 JUL 2015

1. Restriction enzyme cut was designed for verifying the following BioBricks,

|  | Tubes | Cutting (3.1) | Expected fragment size |
| --- | --- | --- | --- |
| BBa\_B0015 | x3 | NotI/ PstI | 163/2036 |
| BBa\_R0051 | x3 | XhoI/ PstI | 114/2005 |
| BBa\_C0051 | x3 | NotI | 800/2000 |
| BBa\_K1218011 | x3 | NotI/ PstI | 5000/2000 |
| BBa\_K577881 | x3 | NotI/ BamHI | 1200/3000 |

2. The fragment size of the above BioBricks was expected to be confirmed by running 1% agarose gel in syBRsafe and TAE buffer but the bands were gone too far before turning off Electrophoresis Power Supply.

[##### Top](#Top)

#### Week 4

## 13 JUL 2015

1. Restriction enzyme cut was designed again for verifying the following BioBricks,

|  | Tubes | Cutting | Expected fragment size |
| --- | --- | --- | --- |
| BBa\_B0015 | x3 | EcoRI / PstI (2.1) | 163/2036 |
| BBa\_R0051 | x3 | EcoRI / PstI (2.1) | 90/2000 |
| BBa\_B0030 | x3 | EcoRI / PstI (2.1) | 56/2000 |
| BBa\_K588001 | x3 | EcoRI / PstI (2.1) | 90/2000 |
| BBa\_C0051 | x3 | NotI (3.1) | 800/2000 |
| BBa\_K1218011 | x3 | EcoRI / BamHI (2.1) | 4000/3100 |
| BBa\_K577881 | x3 | EcoRI / BamHI (2.1) | 1200/3000 |

2. The fragment size of the above BioBricks was confirmed by running 1% agarose gel for the first four BioBricks and 3% agarose gel for the latter three in syBRsafe and TAE buffer,

→ in 1% agarose gel,

- BBa\_C0051 (i) and (ii) showed problematic band size

- BBa\_C0051 (iii), BBa\_K1218011 (i), (ii), (iii) and BBa\_K577881 (i), (ii), (iii) showed
fragments with expected size

→ in 3% agarose gel,

- BBa\_R0051 (i), (ii), (iii) did not come up with expected fragments

- BBa\_B0015 (i), (ii), (iii) showed fragments with expected size

- BBa\_B0030 (i), (ii), (iii) and BBa\_K588001 (i), (ii), (iii) did show expected results but
the bands were not clear to be seen

## 14 JUL 2015

1. Restriction enzyme cut was designed for verifying the following BioBricks,

|  | Tubes | Cutting | Expected fragment size |
| --- | --- | --- | --- |
| BBa\_R0051 | x3 | NcoI / EcoRI (2.1) | 749/1370 |
| BBa\_B0030 | x3 | NcoI / EcoRI (2.1) | 749/1336 |
| BBa\_K588001 | x3 | NcoI / EcoRI (2.1) | 749/1370 |

2. The fragment size of the above BioBricks was confirmed by running 3% agarose gel in syBRsafe and TAE buffer,

- BBa\_R0051 (i), (ii), (iii) showed problematic band size

- BBa\_B0030 (i), (ii), (iii) showed expected result

- BBa\_K588001 (i), (ii), (iii) showed an extra band

## 15 JUL 2015

1. Restriction enzyme cut was re-designed for verifying the following BioBricks again,

|  | Tubes | Cutting | Expected fragment size |
| --- | --- | --- | --- |
| BBa\_R0051 | x4 | NcoI / PstI (3.1) | 805/1280 |
| BBa\_B0030 | x4 | NcoI / PstI (3.1) | 839/1280 |
| BBa\_K588001 | x4 | NcoI / PstI (3.1) | 839/1280 |

2. The fragment size of the above BioBricks was confirmed by running 1% agarose gel in syBRsafe and TAE buffer,

- BBa\_R0051 (i), (ii), (iii) still showed problematic band size

- BBa\_B0030 (i), (ii), (iii) showed expected result

- BBa\_K588001 (i), (ii), (iii) still showed problematic band size and extra bands

3. BBa\_C0051 (iii) was transformed again using DH5β competent cells borrowed on CM30 plate to amplifying successful plasmid as (i) and (ii) could not be verified from the gel result.

4. BBa\_I13973 was resuspended from the kit and stored in an labelled Eppendorf tube.

## 16 JUL 2015

1. Transformation result of BBa\_C0051 (iii) was confirmed that >30 colonies could be found on CM30 plate.

2. The following BioBricks were transformed again using DH5β competent cells borrowed on CM30 plates to amplify successfully verified plasmids,

- BBa\_K1218011 (iii)

- BBa\_K577881 (iii)

- BBa\_B0030 (i)

- BBa\_B0015 (iii),

3. Meanwhile, BBa\_I13973 resuspended on the previous day was firstly transformed using borrowed DH5β competent cells on CM30 plate.

## 17 JUL 2015

1. Transformation result was confirmed that

- BBa\_K577881 (iii), BBa\_B0030 (i) and BBa\_B0015 (iii) showed >30 colonies

- BBa\_K1218011 (iii) showed several small colonies

- BBa\_I13973 showed no colonies on the plate

2. 11 LB plates with CM30 were prepared.

3. We just realized some of the BioBricks were ampicillin resistant rather than chloramphenicol resistant, so transformation of the following BioBricks was repeated using DH5β competent cells borrowed but on Amp100 plates,

- BBa\_R0051

- BBa\_J61100 from first resuspension

- BBa\_J61100 from second resuspension

4. At the same time, BBa\_I13973 and BBa\_K1218011 were transformed using DH5β competent cells borrowed on CM30 plates.

[##### Top](#Top)

#### Week 5

## 20 JUL 2015

1. Transformation result was confirmed that

- BBa\_R0051 showed no colonies

- BBa\_J61100 from first resuspension showed 23 colonies

- BBa\_J61100 from second resuspension showed no colonies

- BBa\_I13973 showed no colonies

- BBa\_K1218011 showed no colonies

2. BBa\_I13973 and BBa\_K1218011 were transformed using DH5β competent cells borrowed on CM30 plates again.

3. New BioBricks we requested on 4 Jul arrived and were streaked on plates,

- BBa\_J61100 (agar) on Amp100 plate

- BBa\_K512001 (agar) on CM30 plate

- BBa\_K588000 (agar) on CM30 plate [the twin of BBa\_J119124 that we requested]

- BBa\_K786031 (agar) on CM30 plate

- BBa\_K786032 (agar) on CM30 plate

## 21 JUL 2015

1. Transformation result was confirmed that

- BBa\_I13973 showed no colonies

- BBa\_K1218011 showed no colonies

- BBa\_J61100 (agar): TMTC (too many to count)

- BBa\_K512001 (agar): TMTC

- BBa\_K588000 (agar): TMTC

- BBa\_K786031 (agar): TMTC

- BBa\_K786032 (agar): TMTC

2. The following BioBricks were transformed using DH5β competent cells borrowed on both CM30 plates and Amp100 plates

- BBa\_I13973

- BBa\_K1218011

3. Colonies from the following plates were picked and incubated overnight,

- BBa\_J61100 (agar) on Amp100 plate

- BBa\_K588000 (agar) on CM30 plate

- BBa\_K512001 (agar) on CM30 plate

- BBa\_K786031 (agar) on CM30 plate

- BBa\_K786032 (agar) on CM30 plate

## 22 JUL 2015

1. Transformation result was confirmed that

- BBa\_I13973 showed no colonies on both CM30 and Amp100 plates

- BBa\_K1218011 showed no colonies on both CM30 and Amp100 plates

2. 12 LB plates with CM30 were prepared.

3. The following BioBricks were miniprepped,

- BBa\_J61100

- BBa\_K786032

- BBa\_K512001

- BBa\_K786031

- BBa\_K588000

## 23 JUL 2015

1. DNA concentration of the following BioBricks was measured by GeneQuant

|  |  |
| --- | --- |
| BBa\_J61100 (i) | 0.107 au → 267.5 ng/uL |
| BBa\_J61100 (ii) | 0.104 au → 260 ng/uL |
| BBa\_K786032 (i) | 0.169 au → 422.5 ng.uL |
| BBa\_K786032 (ii) | 0.156 au → 390 ng/uL |
| BBa\_K512001 (i) | 0.168 au → 422.5 ng/uL |
| BBa\_K512001 (ii) | 0.164 au → 410 ng/uL |
| BBa\_K786031 (i) | 0.205 au → 512.5 ng/uL |
| BBa\_K786031 (ii) | 0.235 au → 587.5 ng/uL |
| BBa\_K588000 (i) | 0.138 au → 345 ng/uL |
| BBa\_K588000 (ii) | 0.141 au → 352.5 ng/uL |

Restriction enzyme cut was designed to verify the following BioBricks

|  | Tubes | Cutting | Expected fragment size |
| --- | --- | --- | --- |
| BBa\_J61100 | x2 | ApaLI (2.1) | 1246/845 |
| BBa\_K588000 | x2 | NotI (3.1) | 351/2046 |
| BBa\_K512001 | x2 | BamHI, NcoI (3.1) | 311/793/897/2387/3201 |
| BBa\_K786031 | x2 | NotI (3.1) | 980/2046 |
| BBa\_K786032 | x2 | NotI (3.1) | 2691/2046 |

3. The fragment size was confirmed by running 1% agarose gel in syBRsafe and TAE buffer

- BBa\_J61100, BBa\_K786032 and BBa\_K588000 showed expected results

- BBa\_K512001 and BBa\_K786031 showed wrong number of bands

4. BBa\_K1333315 and BBa\_I13504 were resuspended from the kit and transformed using DH5β competent cells borrowed on CM30 plates.

5. Requests to Headquarter were made for the following BioBricks

- BBa\_K1218011

- BBa\_I13973

- BBa\_Q04510

## 24 JUL 2015

1. Transformation result was confirmed that both BBa\_K1333315 and BBa\_I13504 showed >10 small colonies.

2. Restriction enzyme cut was re-designed to verify the following BioBricks again

|  | Tubes | Cutting | Expected fragment size |
| --- | --- | --- | --- |
| BBa\_K512001 | x2 | PstI (3.1) | 4332/2138/1169 |
| BBa\_K786001 | x2 | NotI (3.1) | 980/2046 |

3. The fragment size was confirmed by running 0.7% agarose gel in syBRsafe and TAE buffer that both BBa\_K512001 and BBa\_K786031 showed wrong band size.

[##### Top](#Top)

#### Week 6

## 27 JUL 2015

1. Colonies of BBa\_I13504 and BBa\_K1333315 were picked from plates and incubated overnight.

## 28 JUL 2015

1. BBa\_I13504 and BBa\_K1333315 were miniprepped.

2. DNA concentration of the following BioBricks was measured by Nanodrop 1000

|  |  |
| --- | --- |
| BBa\_I13504 (i) | 69.9 ng/uL |
| BBa\_I13504 (ii) | 83.0 ng/uL |
| BBa\_I13504 (iii) | 74.8 ng/uL |
| BBa\_K1333315 (i) | 99.5 ng/uL |
| BBa\_K1333315 (ii) | 112.6 ng/uL |
| BBa\_K1333315 (iii) | 100.4 ng/uL |

3. Restriction enzyme cut was designed to verify the following BioBricks

|  | Tubes | Cutting | Expected fragment size |
| --- | --- | --- | --- |
| BBa\_I13504 | x3 | NotI (3.1) | 899/2046 |
| BBa\_K1333315 | x3 | NotI (3.1) | 1276/2046 |

4. Fragment size was confirmed by running 0.7% agarose gel in syBRsafe and TAE buffer that both BBa\_I13504 and BBa\_K1333315 showed expected result.

## 29 JUL 2015

1. A new MG1655 plate was streaked for making new competent cells.

## 30 JUL 2015

1. 200 mL of LB was prepared for making new competent cells.

2. A colony from MG1655 plate was picked and incubated for making new competent cells.

3. The following BioBricks requested from Headquarter were arrived and streaked on CM30 plates

- BBa\_K1218011 (agar)

- BBa\_I13973 (agar)

[BBa\_Q04510 was not available at HQ]

## 31 JUL 2015

1. Both BBa\_K1218011 and BBa\_I13973 plates showed >30 colonies.

2. New MG1655 competent cells were prepared.

[##### Top](#Top)

#### Week 7

## 03 AUG 2015

1. MG1655 competent cells were tested with the Competent Cell Test Kit.

2. Colonies of BBa\_I13973 and BBa\_K1218011 were picked from the plates and incubated overnight.

## 04 AUG 2015

1. Competency test result was confirmed that MG1655 competent cells prepared on 31 Jul failed as no colonies could be found on any plates.

2. BBa\_I13973 and BBa\_K1218011 were miniprepped and their DNA concentration was measured by Nanodrop 2000,

|  |  |
| --- | --- |
| BBa\_I13973 | 81.3 ng/uL |
| BBa\_K1218011 | 250.5 ng/uL |

3. Restriction enzyme cut was designed for verifying the following BioBricks

|  | Tubes | Cutting | Expected fragment size |
| --- | --- | --- | --- |
| BBa\_I13973 | x1 | BamHI, NotI (3.1) | 998/2046 |
| BBa\_K1218011 | x1 | NotI (3.1) | 3935/2046/1169 |

4. Fragment size was confirmed by running 0.7% agarose gel in syBRsafe and TAE buffer that both BBa\_K1218011 and BBa\_I13973 showed expected results.

## 05 AUG 2015

1. 100 mL of 50mM CaCl2 and 100 mL of 50mM CaCl2/15% glycerol were prepared for making competent cells.

## 06 AUG 2015

1. 500 mL of LB was prepared for making competent cells.

2. A colony was picked from the MG1655 plate streaked on 29 Jul and incubated overnight for making competent cells.

## 07 AUG 2015

1. New MG1655 competent cells were prepared.

[##### Top](#Top)

#### Week 8

## 11 AUG 2015

1. 200 mL of LB broth was prepared.

2. 16 LB plates with CM30 were prepared.

3. MG1655 competent cells prepared on 7 Aug were tested with the Competent Cell Test Kit with DH10B as control.

## 12 AUG 2015

1. Competency test result was confirmed that the MG1655 competent cells failed as no colonies could be found on any plates.

2. BBa\_K577881 was transformed into BL21(DE3) competent cells on CM30 plate.

## 13 AUG 2015

1. Transformation result was confirmed after 16.5 hours incubation at 37 degree Celsius that BBa\_K577881 was successfully transformed into BL21(DE3).

2. 1 colony was picked from BBa\_K577881 plate in BL21(DE3) spread on 12 Aug and incubated overnight at 37 degree Celsius.

## 14 AUG 2015

1. GFP signals were tested by microplate reader Varioskan Flash expressed by BBa\_K577881 induced by different concentration of L-arabinose.

| Stock (w/v) | Final Concentration |
| --- | --- |
| -- | 0% |
| 0.02% | 0.0002% |
| 0.2% | 0.002% |
| 1% | 0.01% |
| 2% | 0.02% |
| 5% | 0.05% |
| 10% | 0.1% |
| 20% | 0.2% |
| 50% | 0.5% |

→ Result: no GFP signals

2. BBa\_K577881 was induced by 10 uL 1% arabinose on CM30 plate and incubated overnight at 37 degree Celsius.

[##### Top](#Top)

#### Week 9

## 17 AUG 2015

1. The plate spread on 14 Aug showed no GFP signal.

2. 200 mL of LB broth was prepared.

3. 1 colony was picked from BBa\_K577881 plate in BL21(DE3) spread on 12 Aug and incubated overnight at 37 degree Celsius.

4. 20% and 50% arabinose solution were prepared.

5. BBa\_K577881 was induced by 50 uL 20% arabinose on CM30 plate and incubated overnight at 37 degree Celsius.

## 18 AUG 2015

1. The plate spread on 17 Aug showed no GFP signal.

2. GFP signals were tested by microplate reader Varioskan Flash expressed by BBa\_K577881 induced by different concentration of L-arabinose.

| Stock (w/v) | Final Concentration |
| --- | --- |
| -- | 0% |
| 0.02% | 0.0002% |
| 0.2% | 0.002% |
| 1% | 0.01% |
| 2% | 0.02% |
| 5% | 0.05% |
| 10% | 0.1% |
| 20% | 0.2% |

→ Result: no GFP signals

3. 1 colony was picked from BBa\_K577881 plate in DH10B spread on 16 Jul and incubated overnight at 37 degree Celsius.

## 19 AUG 2015

1. SDS-PAGE was performed to see whether the GFP E0040 was well expressed by BBa\_K577881 induced by arabinose in BL21(DE3).

## 20 AUG 2015

1. 16 CM30 LB agar plates were prepared.

2. BBa\_K577881 was transformed into DH10B competent cells on CM30 plate.

## 21 AUG 2015

1. Transformation result was confirmed that BBa\_K577881 was successfully transformed into DH10B competent cells (TMTC).

[##### Top](#Top)

#### Week 10

## 24 AUG 2015

1. 3 colonies were picked from the CM30 plate of BBa\_K577881 transformed on 20 Aug and incubated overnight at 37 degree Celsius.

## 25 AUG 2015

1. BBa\_K577881 was miniprepped and DNA concentration was measured using Nanodrop 2000.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
||  |  |
| --- | --- |
| BBa\_K577881 (1) | 74.6 ng/uL |
| BBa\_K577881 (2) | 202.5 ng/uL |
| BBa\_K577881 (3) | 85.3 ng/uL |

## 26 AUG 2015

1. 500 mL of SOB was prepared.

2. 1 colony was picked from the CM30 plate of BBa\_K577881 transformed on 20 Aug and incubated overnight at 37 degree Celsius.

3. SDS-PAGE gel was prepared (15% separating gel and 5% stacking gel).

## 27 AUG 2015

1. GFP signals were tested by microplate reader Varioskan Flash expressed by BBa\_K577881 induced by different concentration of L-arabinose.

| Stock (w/v) | Final Concentration |
| --- | --- |
| -- | 0% |
| 0.02% | 0.0002% |
| 0.2% | 0.002% |
| 1% | 0.01% |
| 2% | 0.02% |
| 5% | 0.05% |
| 10% | 0.1% |
| 20% | 0.2% |

→ Result: no GFP signals

2. Primers of gBlock assembly were prepared so that the concentration became 10 uM while gBlock final concentration was 10 ng/uL.

3. PCR was performed but running gel was failed due to human error so 2 pieces of new 1% agarose gel were prepared and PCR was done again overnight.

## 28 AUG 2015

1. To check whether GFP (27 kDa) was expressed in BBa\_K577881 when the BioBrick was induced with 0.2% arabinose,

- SDS-PAGE was performed using Pageruler sm0671 as the marker → Results: a thick band of about 25 kDa after staining with Coomassie blue

- Western blot was performed using Pageruler sm0671 as the marker → Negative results

2. A new CM30 plate was streaked with BBa\_K577881 in DH10B induced by 0.2% arabinose as an alternative method to check for GFP expression.

3. Running gel was performed to confirm the PCR products that gBlock 1, 3, 4 and 5 showed correct band size though gBlock 4 had some non-specific cut. gBlock 2 showed wrong band size. PCR was done again.

4. For gBlock 1, 3, 4 and 5, gel extraction was performed to purify these gBlocks. The concentration was measured by Nanodrop 2000.

|  |  |
| --- | --- |
| gBlock 1 | 7.7 ng/uL |
| gBlock 3 | 9 ng/uL |
| gBlock 4 | 4.4 ng/uL |
| gBlock 5 | 36.5 ng/uL |

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#### Week 11

## 31 AUG 2015

1. Running gel was performed to confirm the PCR products that gBlock 1, 3, 4 and 5 showed correct band size despite of non-specific cuts. gBlock 2 still showed wrong band size.

2. PCR (Tm-5) was done again and after running the gel, gBlock 1 and 5 were fine with expected band size while the expected bands of gBlock 3 and 4 were too vague to be seen. And gBlock 2 was still problematic about its band size.

3. To detect GFP signal expressed by BBa\_K577881 in DH10B induced by 0.2% arabinose,

- by using plate reader Varioskan Flash, the result was unreliable as the maximum RFU was at 501 nm and 511 nm for the well containing BBa\_K577881 overnight culture with arabinose as well as the one with PBS alone

- by using confocal microscope, GFP could be seen from the BBa\_K577881 overnight culture with arabinose

- from the plate streaked on 28 Aug, green fluorescence could be spotted under UV box

4. 1 colony was picked from each BBa\_K577881 plate transformed into DH10B on 20 Aug and BL21(DE3) on 12 Aug, the collected colonies were incubated overnight at 37 degree Celsius.

5. 2 new CM30 plates were streaked with BBa\_K577881 in DH10B and BL21(DE3) induced by 0.2% arabinose to check for GFP expression.

## 01 SEP 2015

1. PCR was performed and its products were confirmed after running the gel. gBlock 1, 3, 4 and 5 were fine with the band size but gBlock 4 was somehow vague to be seen under UV. gBLock 2 showed wrong band size.

2. Gel extraction for purifying gBlock 1, 3 and 5 was failed due to human error.

3. Since gBlock 2 failed every time, the original fragment was tested by running gel. Yet no bands could be seen in the gel photo. 2-step PCR was performed for gBlock 2 and 4.

4. 1-hour arabinose induction was performed for GFP expression by BBa\_K577881.

## 02 SEP 2015

1. The PCR products were confirmed after running the gel that gBlock 2 still showed wrong band size while gBlock 4 was invisible in gel photo.

2. PCR was performed for gBlock 1, 2, 3 and 4, but this time with DMSO added to eliminate any possible secondary structures that may account for gBlock amplification failure. The products were confirmed from the gel that only gBlock 2 showed wrong band size.

3. Gel extraction was performed to purify gBlock 1, 3, and 4. The concentration was measured by Nanodrop 2000,

|  |  |
| --- | --- |
| gBlock 1 | 19.5 ng/uL |
| gBlock 3 | 23.8 ng/uL |
| gBlock 4 | 8.7 ng/uL |

4. gBlock 2 original fragment was tested by running agarose gel to see if it is defective but there was a very thin band of the correct band size.

5. The GFP test performed on 1 Sep was on-going overnight and the GFP was measured by using PerkinElmer Multimode Plate Reader. It showed that RFU generally increased with arabinose concentration and time. Also, the expression of BBa\_K577881 was better in DH10B than in BL21(DE3).

## 04 SEP 2015

1. PCR was performed for gBlock 2 with DMSO added. This time we had tried 2 Ta for the 30-second annealing step. However the 1kb+ ladder and gBlock bands were unclear due to syBRsafe lost during gel piece handling.

2. All gBlock original fragments were tested by running agarose gel. Only gBlock 1, gBlock 3 and gBlock 5 showed expected bands while gBlock 2 still came up with a band of 600 bp and gBlock 4 went invisible.

3. gBlock 2 and gBlock 4 original fragments were tested again by running the agarose gel but no bands were found for both gBlocks.

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#### Week 12

## 07 SEP 2015

1. gBlock 2 original fragment was tested by running agarose gel but still no bands were observed.

2. HiFi assembly was performed to assemble all 5 gBlocks (PCR products of each gBlock, except gBlock 2 which we used its stock) with the backbone pSB1C3, along with a positive control. Both products were transformed into DH10B competent cells on CM30 plates with 10 uL of 20% arabinose stock and 10 uL of 0.1% tryptophan stock added.

## 08 SEP 2015

1. Transformation results of HiFi products with the positive control were confirmed that only the positive control showed >30 colonies while HiFi product transformation failed even with arabinose and tryptophan added.

2. HiFi products were tested by running agarose gel and several bands instead of one single band were observed.

3. Using the same HiFi products, transformation into DH10B competent cells was performed again under the same setting but this time 0.02% arabinose and 0.001% tryptophan were added during the recovery step.

4. The PCR product of gBlock 4 was amplified by PCR and the products were stored overnight at 4 degree Celsius.

## 09 SEP 2015

1. Second transformation results of HiFi products were confirmed that 14 small pink colonies were found.

2. The PCR gBlock 4 overnight products were collected and tested by running agarose gel but no bands were observed.

3. PCR was performed for gBlock 2 but this time we used Taq polymerase with dNTPs. Ta 53.9 degree Celsius was used for the 30-second annealing step. Yet, no bands were observed after running the product on an agarose gel.

4. HiFi assembly was performed again but this time we used the stock of both gBlock 2 and gBlock 4 with the PCR products of gBlock 1, gBlock 3 and gBlock 5 and pSB1C3. The HiFi product was transformed into Dh10B competent cells with 0.02% arabinose and 0.001% tryptophan added on a CM30 plate.

5. 8 colonies were picked from the CM30 plate with HiFi product in DH10B and incubated in LB-Ara-Trp-CM30 overnight.

## 10 SEP 2015

1. Third transformation results of HiFi products were confirmed that no colonies were found.

2. The HiFi products from second transformation were miniprepped.

## 11 SEP 2015

1. The concentration of the miniprepped HiFi products was measured by using Nanodrop 2000.

|  |  |
| --- | --- |
| HiFi product (1) | 190.5 ng/uL |
| HiFi product (2) | 202.8 ng/uL |
| HiFi product (3) | 224.8 ng/uL |
| HiFi product (4) | 211.4 ng/uL |
| HiFi product (5) | 233.1 ng/uL |
| HiFi product (6) | 227.7 ng/uL |
| HiFi product (7) | 231.8 ng/uL |
| HiFi product (8) | 256.5 ng/uL |

2. All miniprepped HiFi products were tested by running a 0.7% agarose gel. But each product showed 2 bands of 2000 bp and 3000 bp instead of just a single band of around 9000 bp.

3. 2-step PCR was performed using Taq polymerase to amplify both gBlock 2 and the whole construct from the miniprep HiFi product (8).

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#### Week 13

## 14 SEP 2015

1. The 2-step PCR products were tested by running a 0.7% agarose gel. However, the band sizes of both gBlock 2 and the whole construct were wrong though they showed similar bands.

2. New gBlock 2 had arrived and resuspension with TE buffer was done so that the final concentration became 10 ng/uL. The fragment was then tested by running a 0.7% agarose gel but the band was invisible due to the low concentration of gBlock 2 in the well.

3. PCR was performed to amplify the new gBlock 2 using Q5 polymerase. 2 temperatures were used for the 30-second annealing step: 59.9 degree Celsius and 61.5 degree Celsius.

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