iGEM 2015 — Team Pitt

# Team:Pitt/Notebook

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| [Home](https://2015.igem.org/Team%3APitt) | [Project](https://2015.igem.org/Team%3APitt/Description)  * [Overview](https://2015.igem.org/Team%3APitt/Description) * [Estrogen Sensor](https://2015.igem.org/Team%3APitt/Estrogen/Project) * [Protease Sensor](https://2015.igem.org/Team%3APitt/Protease/Project) * [Three-Hybrid Versatile Sensor](https://2015.igem.org/Team%3APitt/3-hybrid/Project) * [Amplification and Quenching](https://2015.igem.org/Team%3APitt/Amplification/Project) | [Notebook](https://2015.igem.org/Team%3APitt/Notebook)  * [Sensor Extract Protocol](https://2015.igem.org/Team%3APitt/Protocols) * [Daily Notebook](https://2015.igem.org/Team%3APitt/Notebook) | [Parts](https://2015.igem.org/Team%3APitt/Parts) | [Human Practices](https://2015.igem.org/Team%3APitt/Practices) | [Team](https://2015.igem.org/Team%3APitt/Team)  * [Meet us!](https://2015.igem.org/Team%3APitt/Team) * [Attributions](https://2015.igem.org/Team%3APitt/Attributions) | [Sponsors](https://2015.igem.org/Team%3APitt/Sponsors) | [2015 iGEM Home](https://igem.org/Main_Page) |

## Notebook

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*Note: all experiments were done with all members present, unless otherwise noted*

#### June 15, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Transformation of plasmids from Pardee paper | Into DH5a, all transformations successful |
| Grow O/N of DH5a | Competent cell prep (Konstantin Borisov) |

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#### June 16, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| O/N of transformations | Miniprep |
| DH5a competent cell prep | 25 tubes of 200ul each, enough to last all summer! (Konstantin Borisov, help from Tatyana Yatsenko) |

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#### June 17, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Transformation of plasmids from iGEM registry | Into new DH5a, all transformations successful |

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#### June 18, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Test PT7 lysate with Pardee paper plasmids | No convenient method of visualization, so we started looking for a plate reader |

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#### June 22, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| PCR pSB1C3 with backbone fwd and rev primers | For Gibson assembly, learning process |
| Gel of PCR products | Visualization and purification of pSB1C3 backbone |
| Gel purification | Obtained enough backbone for the rest of the summer! |

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#### June 23, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Gibson assembly of protease parts from IDT, transformation into DH5a | All transformations multiple colonies |
| Transformation of CMU ERT7 into DH5a | Success, faint yellow from YFP seen under blue light |

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#### June 24, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Miniprep and diagnostic gel of Gibson assembly | All 3 had appropriate bands |
| Crude lysate preparation from ERT7/DH5a | Original S30 extract protocol, much too dilute due to incorrect weighing |

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#### June 25, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Sequencing of Gibson'd protease constructs | 5/6 had correct sequence, other one had significant deletion |
| ERT7 lysate tests on Tecan plate reader | No results because of high dilution |

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#### June 29, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Repetition of Pardee experiments | First success! |
| Freeze-dry constructs on paper | Test validity of Pardee results (Tatyana Yatsenko, Garrett Green) |

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#### June 30, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Grow O/N of NiCo21(DE3) | Competent cell prep (Konstantin Borisov) |
| Test freeze-dried constructs | No results on Tecan reader |

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#### July 1, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| NiCo21(DE3) competent cell prep | 20 tubes of 250ul each (Konstantin Borisov) |
| Solution tests of Pardee toehold sensors | No results |

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#### July 6, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Restocked solutions, plates and media | Running low on supplies |

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#### July 7, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Transform plasmids for ERT7: wildtype T7, T7-YFP, T7 with sites | Controls for estrogen project, into DH5a |

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#### July 9, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Created cell lysates from NiCo21(DE3), NiCo21(DE3) with IPTG induction, DH5a, DH5a with ERT7-YRP | Lots of controls |

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#### July 10, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Set up PCR for vector backbone | Discovered original vector backbone had been left out and degraded |
| Prepared glycerol stocks of protease constructs | In NiCo21(DE3) cells |

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#### July 11, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Gel electrophoresis on PCR | Gel was run in the wrong direction... lost all DNA |

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#### July 13, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Another PCR of vector backbone and Gel | Success! 3 lanes of vector backbone extracted. |

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#### July 16, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Gibson of MMP9 fusion and protease reporter, transformation into DH5a | all transformations had a lot of colonies |

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#### July 17, 2015

| Protocol | Notes/results/purpose |
| --- | --- |
| Diagnostic gel of all DNA made | Cut with EcoRI, SpeI (Konstantin Borisov) |

![](https://static.igem.org/mediawiki/2015/c/cb/Prettygelpitt.png)

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