Week 26 (June 26 - July 2)

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• Wet Lab Overview

  Ordered primers, checked cell stocks and prepared LB plates

Week 27 (July 3 - 9)

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• 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

  Inoculation in liquid culture of bacterial stub shipped by Addgene with plasmids 3xFLAG-pdCas9 (in LB-Chloramphenicol (CAM)) and pgRNA-bacteria (in LB-Ampicillin (AMP)) Creation of glycerol stock (stored at -80 °C)

  

  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)

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• 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

  3xFLAG-pdCas9 and pgRNA plasmid purification, linearization with restriction enzymes (RE) and size control via Gel electrophoresis (GE)

  

  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)

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• 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

  Insertion of gRNA seed sequences via PCR. pgRNA1, pgRNA2, pgRNA3 are obtained. Transformation of E. coli mach1 with pgRNA1, pgRNA2, pgRNA3 (Mix&ampGo kit) Plasmids purification and control via GE

  

  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)

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• Wet Lab Overview

  Making first transformations in all subprojects.

  

  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

  Plasmids purification from E. coli DH5-α transformed cell. Size control via GE Re-inoculation of E. coli DH5-α in fresh LB broth with proper antibiotic(s) Growth curve OD₆₀₀ of E. coli DH5-α single and double transformed

  

  Protein Import

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

Week 31 (July 31 - August 6)

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• Wet Lab Overview

  

  Number Control

  Creation of glycerol stock of E. coli DH5-α transformed cells. Plating of E. coli DH5-α onto LB agar with proper antibiotic(s) OD₆₀₀ growth curve Plating serial dilution of E. coli DH5-α transformed cells for Colony Forming Unit (CFU) counting

  

  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 pET102 vector, and miniprep to extract the vector.

Week 32 (August 7 - 13)

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• Wet Lab Overview

  

  Number Control

  Growth curve via CFU enumeration

  

  Protein Import

  Verification of pET102 iGEM with digestion and sequencing.

Week 33 (August 14 - 20)

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• Wet Lab Overview

  

  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

  Creation of 50 mL (250 mL flasks) liquid culture of E. coli DH5-α transformed cells (two for each strain) OD₆₀₀ growth curve with and without dCas9 expression inducer (Tetracycline)

  

  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)

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• Wet Lab Overview

  

  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

  OD₆₀₀ growth curve with and without dCas9 expression inducer (Tetracycline)

  

  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)

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• Wet Lab Overview

  

  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

  Creation of empty 3xFLAG-pdCas plasmid via double digestion (named EpdCas9). Transformation of E. coli DH5-α with EpdCas9, pgRNA-bacteria (that is, without seed sequence) and double transformation with both plasmids. Only E. coli DH5-α EpdCas9 grew

  

  Protein Import

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

Week 36 (September 4-10)

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• Wet Lab Overview

  

  Interdependency

  TrpE and YddG sequenced: TrpE correct, YddG incorrect. YddG and AroG transformations redone. Successfull first test of growth of E.coli in YNB media pH 7. 1st serial growth experiment in YNB+trp: yeast can grow after E.coli has been removed from the media!

  

  Number Control

  Double transformation of E. coli DH5-α with 3xFLAG-pdCas9 and pgRNA-bacteria. Liquid culture of E. coli DH5-α:

  • 3xFLAG-pdCas9 and pgRNA-bacteria;
  • EpdCas9;
  • 3xFLAG-pdCas9 and pgRNA1;
  • 3xFLAG-pdCas9 and pgRNA2;
  • 3xFLAG-pdCas9 and pgRNA3.

  Transformation control via plasmids purification and RE liniearization and GE New growth protocol was designed: measurement time point every 4-6 hours interspaced with 10x dilution to lower the effect of non-replicating enlarged cells Growth curve using new protocol. dCas9 expression was induced with 800ng/mL Tet. No significant difference observed

  

  Protein Import

  YFP miniprepped. BFP inserted in vector (USER ligation) and transformed into Mach1 cells. CPP vector formation was tricky. Tried different template concentrations, annealing temps, buffers, before we realised that one of the CPP primers had 70% GC content: Successfull PCR after changing to GC buffer. Gel extraction, DpnI digestion to remove template. Blunt end ligation to finish the vector creation. Linearization (opening the USER casette), and insertion of YFP and BFP. Transformed in Mach1 cells.

Week 37 (September 11 - 17)

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• Wet Lab Overview

  

  Interdependency

  2nd serial growth experiment with TrpE and AroG transformants in YNB-trp. Sequencing of YddG and AroG Still no luck in YddG amplification

  

  Number Control

  Change of dCas9 inducer: Anhydrotetracycline (aTc) due to its higher activity. This will lower the growth inhibition effect of high Tet concentration. Growth curve via new protocol and using aTc for dCas9 induction. No significant difference was observed Working strains plasmids purification and control via GE and sequencing (Macrogen commercial service)

  

  Protein Import

  Plasmid extraction of CPP-BFP. Digestion, colony PCR, amplifications and inoculations. Not succesfull. New CPP-vector was made (PCR, gel exctraction, DpnI digestion, ligation) Purified in column instead of gel extraction.

Week 38 (September 18 - 24)

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• Wet Lab Overview

  

  Interdependency

  3rd serial growth experiment in YNB-trp (all week): BL21 transformed with TrpE, AroG and empty vector grown ON. Insertion of TrpE and AroG in biobrick submission vector. Ordered synthetic, codon optimised YddG gene.

  

  Number Control

  Growth curve via new protocol and using aTc for dCas9 induction. No significant difference was observed

  

  Protein Import

  Insertion of YFP and BFP into biobrick submission vector. CPP vector continues. Problematicto assemble.

Week 39 (September 25 - October 1)

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• Wet Lab Overview

  Biobricks sent for sequencing: YFP, BFP, AroG, TrpE.

  

  Interdependency

  4th serial growth experiment in YNB-trp: AroG, TrpE, empty pET102. No yeast growth. Work on TrpE, AroG Biobricks. Received and transformed with synthetic YddG-stop and YddG-his.

  

  Protein Import

  Inoculation of YFP and BFP Biobricks

Week 40 (October 2 - 8)

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• Wet Lab Overview

  Biobricks of YFP, BFP, TrpE and AroG.

  

  Interdependency

  5th Serial growth experiment with TrpE, AroG and Control. Miniprepped and digested YddG-his and YddG-stop, sequenced. Failed SDS page and Western of TrpE, AroG and Empty vector. Induced with 0.5 mM IPTG. Multi-gene constructs: All parts for combining into constructs amplified, started insertion in vector.

  

  Protein Import

  CPP vector finished and linearized. Failed SDS page and Western of YFP and BFP: Induced with 0.1 mM IPTG.

Week 41 (October 9 - 15)

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• Wet Lab Overview

  

  Interdependency

  Multi-gene constructs: Insertion in vector, digestion. Tricky, so ligations and transformations with YddG-AroG-his and YddG-TrpE-AroG, YddG-TrpE-AroG-his redone several times. YddG biobrick created. Continued work on YddG-his (new primer designed with no stop codon in the overhang) Biobrick AroG and TrpE sequenced. 6th serial growth experiment with TrpE, AroG, YddG (his). Low yeast growth in spent media from AroG, growth after TrpE and control are similar.

  

  Number Control

  Thawing and inoculation of E. coli DH5-α 3xFLAG-pdCas9 pgRNA1; 3xFLAG-pdCas9 pgRNA2; 3xFLAG-pdCas9 pgRNA3 glycerol stocks in fresh LB-AMP-CAM broth.

  

  Protein Import

  CPP vector sequenced. Insertion of YFP and BFP in CPP vector. Protein expression analysis and purification of Control, BFP and YFP, done twice. Induced YFP and BFP cells are fluorescing! Transformation and sequencing CPP-YFP and CPP-BFP. Biobrick amplification and sequencing of CPP, CPP-BFP and CPP-YFP. Correct sequence. Initial attempt at insertion in submission vector failed. Repeating expression analysis with varying induction concentrations. Several inoculations and purifications of all variations of CPP and fluorescent protein at multiple levels of inducing agent.

Week 42 (October 16 - 22)

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• Wet Lab Overview

  Insertion of most Biobricks in submission vector.

  

  Interdependency

  Work on multi-gene constructs continued: sequencing, transformations, purifications.... 7th serial growth exp; Control, TrpE, AroG and YddG. 8th serial growth exp, with HPLC-MSmeasurements: with single and multi-gene constructs, and three levels of induction agent. Western blot performed to quantify expression. Work on Biobrick insertion in submission vector, having trouble with restrictions and insertions. Ran out of linearized submission vector: transformed cells with empty vector, extracted and re-linearized it.

  

  Number Control

  OD₆₀₀ growth curve without dilution (old protocol) of E. coli DH5-α

  • 3xFLAG-pdCas9 and pgRNA-bacteria;
  • 3xFLAG-pdCas9 and pgRNA1;
  • 3xFLAG-pdCas9 and pgRNA2;
  • 3xFLAG-pdCas9 and pgRNA3.

  No significant difference was observed. PCR amplification, gel extraction of gRNA 3,5,8 with Biobrick primers.

  

  Protein Import

  Expression (western blot) performed on transformed cells, several attempts. Biobricks restriction and transformation in Mach1. We realized now, that we have used AMP, not CAM as selection for Biobricks, which is why we have had trouble growing CAM resistant transformants.

Week 43 (October 23 - 29)

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• Wet Lab Overview

  Creation of last biobricks, and submission. HPLC-MS quantifications of produced tryptophan.

  

  Interdependency

  Continued work on creating biobricks of triple constructs (YddG-TrpE-AroG with and without His). Submission of biobricks. Final serial growth experiment, with measurements of OD600 and HPLC-MS measurements of tryptophan production.

  

  Number Control

  Termination of number control sub-project.

  

  Protein Import

  Fluorescence microscopy performed on YFP and BFP transformants, with and without CPP: All are fluorescing.Initial attempt to investigate import into onion cells - no success, possibly due to degraded proteins.

Week 44 (October 30 - November 1)

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• Wet Lab Overview

  Last tests of CPP tag and import of fluorescent proteins in multiple species of bacteria.