iGEM 2013 — Team Arizona_State

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# Team:Arizona State/Notebook

### From 2013.igem.org

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* Project »
  + [Home](https://2013.igem.org/Team%3AArizona_State)
  + [Chassis](https://2013.igem.org/Team%3AArizona_State/Nissle)+ [Antigen:MHC Class I Presentation](https://2013.igem.org/Team%3AArizona_State/MHC)
    + [Adjuvants](https://2013.igem.org/Team%3AArizona_State/Adjuvants)
    + [Notebook](https://2013.igem.org/Team%3AArizona_State/Notebook)
* Team »
  + [Members](https://2013.igem.org/Team%3AArizona_State/Team)
  + [Attributions](https://2013.igem.org/Team%3AArizona_State/Attributions)
  + [Official Team Profile](https://igem.org/Team.cgi?id=1190)
* Results »
  + [Data](https://2013.igem.org/Team%3AArizona_State/Data)
  + [BioBricks](https://2013.igem.org/Team%3AArizona_State/Parts)
* Human Practices »
  + [Safety](https://2013.igem.org/Team%3AArizona_State/Safety)
  + [Biosecurity and Law](https://2013.igem.org/Team%3AArizona_State/Biosecurity)
  + [Biosafety Engineering](https://2013.igem.org/Team%3AArizona_State/BSE)
  + [Translational Research](https://2013.igem.org/Team%3AArizona_State/food)
  + [Expert Feedback](https://2013.igem.org/Team%3AArizona_State/IS)
* Extras »
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  + [Future Directions](https://2013.igem.org/Team%3AArizona_State/Future)
  + [References](https://2013.igem.org/Team%3AArizona_State/References)

## May: Week 1

* First ASU iGEM 2013 with new team members
* Introduction to the competition, registration logistics, summer schedules
* Idea Brainstorming
* Review of biobrick cloning, Golden Gate assembly, safety training

## Week 2

* Idea Brainstorming--narrowed down to two project ideas

#### Safety Training:

* Biosafety and Bloodborne Pathogens
* Lab Safety
* Fire Safety
* Recombinant DNA Safety
* Hazardous Waste Management

## June: Week 3

* Started making amp plates in preparation for experimentation

## Week 4

* Started growing up colonies of E. coli K12 MG1655, BL21 (DE3),and N10 B strains along with glycerol stocks
* Performed a restriction, ligation and transformation of the three plasmids and grew them up on a plate
* No colony growth of any of the plasmids

## Week 5

* Performed a PCR amplification of the cadA promoter along with a gel screen that found the band at the appropriate band length
* Transformed and plated both J61100 and E1010 with no growth on either plates
* Gel screen of yebF without the stop codon never showed up on the gel. Ran transformation again.
* Restricted and ligated cadA and BN promoters

## Week 6

* The gel screen of yebF without the stop codon was finally successful
* Transformation of INPNC-MCS and RFP control onto N10B cells was successful
* No growth from transformation of E0020, E0030, and E0040
* Created liquid cultures of E0240 and pCadA in LB Chloro.

## July: Week 7

* Performed restriction digest of pCadA, pBN, and YebF
* Performed seven different ligations
* Miniprepped and nanodropped E0240 and pCadA.
* Miniprepped YebF+GMCSF and PelB leader sequence.
* Flow cytometry indicated that there was no GFP
* Transformed J23100 yet no plate growth

## Week 8

* Ordered Antigen primers
* Ligation and transformation of PelB+GMCSF+pSB1C3/4K5
* Miniprep showed no successful ligation and transformation

## Week 9

* PCR of MelanA and Flu M1 worked via confirmation from gel screen
* Nissle would not grow
* Transformed I13522 into N10B on LB Amp

## August: Week 11

* Grew up six cultures of ligations
* Miniprepped the PSB1c3 backbone and grew up additional backbone in cultures
* Transformed the GFP and GMCSF in the backbone
* Ran LLO pcr on gel and all had correct band lengths

## September: Week 15

* Made ligations of FLU+1c3, MelA+1c3, Flu+J04..., MelA+J04...
* Made liquid cultures of non-red colonies from FLU+1c3, MelA+1c3, Flu+J04..., MelA+J04 transformation
* Biobricks made and sent: LLO with promoter RBS, YEb-GMCSF, GMCSF, FLU1, and MelanA

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