iGEM 2015 — Team GeorgiaTech

# Team:GeorgiaTech/Notebook

![](https://static.igem.org/mediawiki/2015/0/0b/Georgia_tech_banner.gif)

[* HOME](https://2015.igem.org/Team%3AGeorgiaTech)
* PROJECT
  [+ Description](https://2015.igem.org/Team%3AGeorgiaTech/Description)
  [+ Background](https://2015.igem.org/Team%3AGeorgiaTech/Background)

  [+ Achievements](https://2015.igem.org/Team%3AGeorgiaTech/Achievements)
* PARTS
  [+ Team Parts](https://2015.igem.org/Team%3AGeorgiaTech/Parts)
* BENCH WORK
  [+ Experiments](https://2015.igem.org/Team%3AGeorgiaTech/Experiments)
  [+ Protocols](https://2015.igem.org/Team%3AGeorgiaTech/Protocols)
  [+ Results](https://2015.igem.org/Team%3AGeorgiaTech/Results)
  [+ Timeline](https://2015.igem.org/Team%3AGeorgiaTech/Notebook)
  [+ Safety](https://2015.igem.org/Team%3AGeorgiaTech/Safety)
* BEYOND THE BENCH
  [+ Collaborations](https://2015.igem.org/Team%3AGeorgiaTech/Collaborations)
  [+ Human Practices](https://2015.igem.org/Team%3AGeorgiaTech/Practices)
* TEAM
  [+ Members](https://2015.igem.org/Team%3AGeorgiaTech/Members)
  [+ Attributions](https://2015.igem.org/Team%3AGeorgiaTech/Attributions)

## Notebook

### May

![](https://static.igem.org/mediawiki/2015/c/c3/Team_GeorgiaTech_VideoSeries.jpg)

### Week 1 (5/11)

* Conducted background research on cloning techniques, phage display, and modern applications of phage display
* Completed necessary safety trainings
* Brainstormed and researched possible project ideas

### Week 2 (5/18)

* Conducted background research on click chemistry, specifically the CuAAC reaction
* Conducted background research on copper-binding proteins including copper chaperones, copper regulators, and tyrosinase.
* Memorized amino acids
* Kellie worked on the website draft from here onward
* Learned how to use Pymol for protein modeling

### Week 3 (5/25)

* Conducted in-depth research on and selected six different copper binding proteins
* Continued memorizing amino acids and were quizzed on them

### June

### Week 4 (6/1)

* Conducted research on mutagenesis techniques, particularly error prone PCR and DNA shuffling

### Week 5 (6/8)

* Prepared diagrams and figures to illustrate key aspects to our presentation

### Week 6 (6/15)

* Prepared a presentation for our graduate advisors
* Continued creating figures

### Week 7 (6/22)

* Presented a preliminary project proposal to our head advisor (PI), M.G. Finn
* Conducted background research on tagging proteins, specifically the His-6 tag.
* Met with a grad student, Zak Costello, as part of our outreach activities
* Brainstormed beyond the bench/human practices ideas

### July

### Week 8 (6/29)

* Reviewed protocols for DNA purification, PCR, DNA transformation, and cell/bacteria culture

### Week 9 (7/6)

* First week in lab!
* Prepared LB media and LB agarose gel
* Practiced plasmid transformation, purification, and characterization

### Week 10 (7/13)

* Hosted a meetup with Lambert High school; shared project ideas, we assisted them in focusing their project on a smaller/more feasible goal and helped them with the first steps of designing their experiment
* Used ApE to map plasmids for our six proteins
* Designed forward and reverse primers for our six proteins
* Prepared for next week’s lab work by reading about the TOPO vector and related protocols.
* Researched error-prone PCR conditions

### Week 11 (7/20)

![](https://static.igem.org/mediawiki/2015/f/f3/Team_GeorgiaTech_Flanking.jpg)

* Primers arrived!
* Used “primer flanking” PCR to add SfiI and NotI restriction sites to our genes while also cloning them out of their pUC57 vectors
* Purified the PCR products using gel purification and analyzed products with the NanoDrop spectrophotometer and with a second gel
* Ligated PCR product to TOPO vectors
* Transformed the TOPO plasmid into DH5a electocompetent cells and plated for overnight growth
* Repeated plasmid transformation and plating after colony growth was unsuccessful
* Cultured some of the transformed cell in LB liquid media (more nurturing growth media as a positive control)
* Unsuccessful colony growth again; repeated TOPO ligation with lower TOPO:Insert ratio
* Plated cells from five (#2, #3, #1, MNK, T60) samples that grew successfully in liquid media in attempt to isolate colonies (in preparation for colony PCR the following day)
* Ultimately achieved colonies for Atx1, plastocyanin, CusF, T60, and T70 ligation/transformations.
* Sent in purified plasmids for sequencing. All sequences were as expected except for Atx1, but we assumed a sequencing error.

### Week 12 (7/27)

* Successfully grew colonies for Ace1, T80, and tyrosinase ligation/transformations. Did not send these for sequencing but assumed they were “correct” sequences.
* Prepared a stock of TOPO vectors for 8 of the 9 proteins (the missing one being MNK) through mini prep protocol, with extra tubes of Tyr, T80, T70, and T60 stocks in preparation for DNA shuffling
* Digested TOPO products using SfiI and NotI restriction enzymes
* Digested pET23b vectors
* Collected and purified digestion products using gel purification
* Ligated digestion products - joined pET23b with our genes
* Transformed ligation products into chemically competent DH5a *E. coli* cells
* Performed TOPO-gene and pET23b digestions again

### August

### Week 13 (8/3)

![](https://static.igem.org/mediawiki/2015/d/da/Team_GeorgiaTech_Expression.jpg)

Expression Testing

* Ligated digestion products from 7/31 (from our second digestion reaction)
* Transformed ligation products into chemically competent BL21 E. coli cells and plated cells for overnight culture
* Inoculated plated colonies for overnight culture
* Inoculated an aliquot of overnight culture for further growth in 5 mL fresh LB
* At log phase of bacterial growth, induced protein expression with IPTG
* After substantial time for protein expression, measured expression with Aglient Protein 80 kit
* Found excellent expression of tyrosinase homologs, but poor expression of Ace1, plastocyanin, and CusF
* Atx1 and MNK were not tested for expression

![](https://static.igem.org/mediawiki/2015/0/0e/Team_GeorgiaTech_Alignment.gif)

Error-prone PCR

* Prepared stock solutions for dNTPs, MgCl2, and MnCl2
* Prepared reaction mixtures and reacted in PCR thermocycler for about 2 hours
* Gel purified EP-PCR products and characterized them through NanoDrop spectrophometery and gel electrophoresis
* TOPO ligation reaction to insert EP-PCR products into TOPO cloning vectors
* Transformed and plated TOPO ligation vectors for overnight growth
* Inoculated 10 individual tyrosinase colonies
* Various EP-PCR reaction mixtures
  + Following protocols C and F in *Evolutionary Methods in Biotechnology*.
    - Only protocol C yielded good EP-PCR products for Atx1, CusF, and Ace1
  + Empirically modified version of protocol C (elevated MnCl2 concentrations)
    - Good EP-PCR products for all genes with error rate (~1%)
  + Using commercially available Green Master Mix
    - Good EP-PCR products for all genes with error rate (~1%)

### Week 14 (8/10)

* Purified plasmids (miniprep) from liquid cultures harboring 10 mutants of EP-PCR TOPO ligation vectors
* Performed PCR to amplify tyrosinase homologues
* Designed iGEM Informational Pamphlet

### Week 15 (8/17)

* Met to discuss how we are going to accomplish the remaining medal requirements
* Outlined a plan for making informational videos about synthetic biology techniques
* Outlined our poster and PowerPoint presentations
* Distributed iGEM Informational Pamphlet to students in biology and chemistry classes
* Scripted and storyboarded iGEM Video Series

### Week 16 (8/24)

* Worked on figures and graphics for our presentation, poster, and Wiki
* Worked on subcloning our genes into pSB1C3 vectors and submitting the BioBrick to the iGEM Parts Registry
* Worked on filming iGEM Video Series

### Bibliography

1. Brakmann, s.; Lindermann, B. F. *Evolutionary Methods in Biotechnology*; WILEY-VCH Verlag GmbH & Co. KGaA: Weinheim, 2004.

### September

### Week 17 (8/31)

* Continued refining our presentation and figures
* Revised content for the Wiki page
* Uploaded iGEM Video Series to YouTube
* Submitted our BioBricks to the iGEM Parts Registry

### Week 18 - 20 (9/7 - 9/21)

* Practiced our presentation in front of mentors from last year’s iGEM team
* Worked on our poster
* Worked on our Wiki page