Team:UCopenhagen/Notebook

N O T E B O O K

Week 26 (June 26 - July 2)

  • Wet Lab Overview

    Ordered primers, checked cell stocks and prepared LB plates



Week 27 (July 3 - 9)

  • Wet Lab Overview

    Making plates, initial transformations, ordered more primers.

    Interdependency

    Made glycerol stock of MG1655. Designed primers: yddG +/- his-tag, and pointmutations in trpE and aroG

    Number Control

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    Protein Import

    Inoculation and creation of glycerol stocks and streaking on LB plates of 8 bacterial cultures: Micrococcus luteus, Staphylococcus epidermis, Bacillus Cereus, Bacillus licheniformis, Serratia marcescens, Pseudomonas putida and Rhizobium meliloti. Preparation of additional LB plates without antibiotica. Creation of competent Mach1 cells and fast transformation with pRSET A to test efficiency.



Week 28 (July 10 - 16)

  • Wet Lab Overview

    Started making plates and initial transformations

    Interdependency

    PCR from MG1655 stock. Amplified aroG #1 and #2 in first attempt, and trpE #2 at a higher annealing temp, then began optimizing the PCR for yddG and trpE#2 using gradient temperatures without success.

    Number Control

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    Protein Import

    Fluorescent microscopy of S. marascens, E. coli and S. epidermis stained with bodipu. Removal of XbaI sites in pRSET A mod, PCR amplification and gel-extraction of the plasmid. Transformation of competent cells with pRSET amod 1, and test of XbaI site removal by digestion. Started work on pRSETjin and pRSETjin CPP vectors from the pRSET vector without XbaI site.



Week 29 (July 17 - 23)

  • Wet Lab Overview

    Started making plates and initial transformations

    Interdependency

    Continued work on PCR optimization of yddG and trpE#1. Continuous attempts with gradient temperatures. Extracted genomic DNA from MG1655 - one where RNAase was forgotten, so we did two. Successfull PCR of yddG-stop and yddG-his. Started amplification of the whole trpE gene.

    Number Control

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    Protein Import

    Continued working on pRSETjin vectors. Attempted to optimize PCR amplification of pRSETjin vector. Adjusted temperatires, template dilutions, tested digestion of unknown bands, retested primers, redid template, tried different templates, template volumes, primer concentrations, annealing temperatures, redoing templates.



Week 30 (July 24 - 30)

  • Wet Lab Overview

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    Interdependency

    Successfull amplification of whole trpE gene from gDNA, after many attempts due to wrong buffers. Amplified and gel-extracted trpE whole gene to be used as template. Unsuccessfull amplification of trpE#1. Got new fusion primers for trpE#1: Finally succes!

    Number Control

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    Protein Import

    Continued working on pRSETjin vectors. Transformation with BFP, YFP and RFP biobricks



Week 31 (July 31 - August 6)

  • Wet Lab Overview

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    Interdependency

    Well deserved vacations were held, so nothing happened this week.

    Number Control

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    Protein Import

    Extraction of BFP, YFP and RFP from transformations. Amplification of pET102 Trx, to remove Trx domain, then amplification, DPN1 digestion of template. Gelextraction of DPN1 digested, then USER cloned (phosphorylated and ligated). Transformation with the new pRSET vector, and miniprep to extract the vector.



Week 32 (August 7 - 13)

  • Wet Lab Overview

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    Interdependency

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    Number Control

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    Protein Import

    Verification of pET102 iGEM with digestion and sequencing.



Week 33 (August 14 - 20)

  • Wet Lab Overview

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    Interdependency

    Amplification of trpE#1 and trpE#2, aroG#1 and aroG#2, yddGhis and yddG-stop with different template dilutions to find the optimal, then amplification of all, and gel-extraction of trpE#1 and #2.

    Number Control

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    Protein Import

    Gelextraction of minipreps to remove chromosomal DNA. Minipreps of inoculated cells with correct vector. Start optimization for BFP and YFP PCR amplifications.



Week 34 (August 21 - 27)

  • Wet Lab Overview

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    Interdependency

    Amplification and gelextraction of yddG (both versions), aroG#1 and aroG#2. First attempt at growing E.coli in minimal yeast medium - no growth.

    Number Control

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    Protein Import

    Linearization of pET102 iGEM. Attempted amplification of pET102 iGEM with CPP primers. In both cases, issues - maybe due to damaged template? Finally managed to linearize the vector. PCR Amplification and gelextraction of YFP



Week 35 (August 28 - September 3)

  • Wet Lab Overview

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    Interdependency

    Transformation with trpE, aroG, yddG and YFP using linearized pET102 iGEM plasmid. Parts #1 and #2 of trpE and aroG used, combining the two parts give the point mutation. Succesfull growth. Miniprep of the transformed DNA, and digestion of plasmids. Initial failure due to low concentration of DNA in the mini-prep samples.

    Number Control

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    Protein Import

    Transformation and amplification of YFP done in interdependency. Amplification and gelextraction of BFP.