iGEM 2017 — Team Missouri_Rolla

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# Team:Missouri Rolla/Notebook

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# Missouri\_Rolla

---

## 30 October 2017

#### **Ryan Baumann**

#### **Start: 5:00PM**

#### **Cloning efficiency testing of new Universal Acceptor Plasmid**

**Purpose:** To test the cloning efficiency of the Universal Acceptor Plasmid after deletion of the unexpected 10 bp region via site directed mutagenesis PCR

#### **Protocol:**

Golden gate assemblies were performed to insert TNTR3 gblock into our universal acceptor plasmid.
Various molar ratios of TNTR3: Universal acceptor plasmid were tested.
Molar ratios of 1:2, 1:1, 2:1 (recommended ratio), 4:1, and 8:1 were tested for the new plasmid. A 2:1 ratio assembly was performed using our original UAP. A negative control assembly was performed with no inserts added.
Golden gate assemblies contained the following:
1:2 Ratio

|  |  |
| --- | --- |
| Component | Volume |
| TNTR3 (17.64 ng) |  |
| New UAP (100ng) | 1 μL |
| 10X Ligase Buffer | 2 μL |
| Ligase | 1 μL |
| BsmBI | 1 μL |
| MilliQ H2O | up to 20μL |

1:1 ratio

|  |  |
| --- | --- |
| Component | Volume |
| TNTR3 (35.28 ng) |  |
| New UAP (100ng) | 1 μL |
| 10X Ligase Buffer | 2 μL |
| Ligase | 1 μL |
| BsmBI | 1 μL |
| MilliQ H2O | up to 20μL |

2:1 Ratio

|  |  |
| --- | --- |
| Component | Volume |
| TNTR3 (70.56 ng) |  |
| New UAP (100ng) | 1 μL |
| 10X Ligase Buffer | 2 μL |
| Ligase | 1 μL |
| BsmBI | 1 μL |
| MilliQ H2O | up to 20μL |

4:1 Ratio

|  |  |
| --- | --- |
| Component | Volume |
| TNTR3 (141.12 ng) |  |
| New UAP (100ng) | 1 μL |
| 10X Ligase Buffer | 2 μL |
| Ligase | 1 μL |
| BsmBI | 1 μL |
| MilliQ H2O | up to 20μL |

8:1 Ratio

|  |  |
| --- | --- |
| Component | Volume |
| TNTR3 (282.24 ng) |  |
| New UAP (100ng) | 1 μL |
| 10X Ligase Buffer | 2 μL |
| Ligase | 1 μL |
| BsmBI | 1 μL |
| MilliQ H2O | up to 20μL |

2:1 Ratio with original UAP

|  |  |
| --- | --- |
| Component | Volume |
| TNTR3 (70.56 ng) |  |
| Original UAP (100ng) | 1 μL |
| 10X Ligase Buffer | 2 μL |
| Ligase | 1 μL |
| BsmBI | 1 μL |
| MilliQ H2O | up to 20μL |

Negative Control

|  |  |
| --- | --- |
| Component | Volume |
| TNTR3 | None |
| UAP | None |
| 10X Ligase Buffer | 2 μL |
| Ligase | 1 μL |
| BsmBI | 1 μL |
| MilliQ H2O | up to 20μL |

All samples were run with the following thermocycler conditions:
– 20 seconds @ 37°C,
– (3 minutes @ 37°C, 4 minutes @ 16°C) X26
– 5 minutes @ 50°C
– 5 minutes @ 80°C
– 5 minutes @ 16°C
Following assembly, 1 uL of each sample was transformed on to Chloramphenicol Blue-White Screening plates following Intact Genomics ig 5-Alpha chemically competent cell High efficiency Transformation protocol. Dillutions of 20% and 5% were plated and spread using sterile glass beads. A positive control transformation was performed using 1 uL of original UAP.
Colonies were incubated at 37C for 30 hours.
Blue and white colonies were counted on all plates.

#### **Stop: 1:45AM**

#### **Results:**

See project page

#### **Next:**

Boston!

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# Team:Missouri Rolla/Experiments

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# Missouri\_Rolla

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

##### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

##### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:Missouri Rolla/Results

[![](https://static.igem.org/mediawiki/2017/c/cb/T--Missouri_Rolla--plants.jpg)](https://2017.igem.org/Team%3AMissouri_Rolla)

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[Project](https://2017.igem.org/Team%3AMissouri_Rolla/Description)
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# Missouri\_Rolla

# Results

We cloned our basic coding sequences and regulatory parts into PhytoBricks, but were unable to assemble them into our final circuit or transform our ill-fated *Arabidopsis* seedlings.
![](https://static.igem.org/mediawiki/2017/9/97/T--Missouri_Rolla--efficiency.png)

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