Team:GeorgiaTech/Notebook

Notebook

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

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

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

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

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

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

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.

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

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

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%)

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

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

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

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