iGEM 2017 — Team UCopenhagen

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# Team:UCopenhagen/Notebook

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* [Project](https://2017.igem.org/Team%3AUCopenhagen/Project)

  [Project description](https://2017.igem.org/Team%3AUCopenhagen/Project)
  [Interdependency](https://2017.igem.org/Team%3AUCopenhagen/Interdependency)
  [Number control](https://2017.igem.org/Team%3AUCopenhagen/Number-Control)
  [Protein import](https://2017.igem.org/Team%3AUCopenhagen/Protein-Import)
  [Results](https://2017.igem.org/Team%3AUCopenhagen/Results)
  [Parts](https://2017.igem.org/Team%3AUCopenhagen/Parts)
  [Modelling](https://2017.igem.org/Team%3AUCopenhagen/Model)
  [Interlab](https://2017.igem.org/Team%3AUCopenhagen/InterLab)
* [Safety & Labnotes](https://2017.igem.org/Team%3AUCopenhagen/Safety)

  [Safety](https://2017.igem.org/Team%3AUCopenhagen/Safety)
  [Notebook](https://2017.igem.org/Team%3AUCopenhagen/Notebook)
  [Protocols](https://2017.igem.org/Team%3AUCopenhagen/Protocols)
  [Improve](https://2017.igem.org/Team%3AUCopenhagen/Improve)
* [People](https://2017.igem.org/Team%3AUCopenhagen/Team)

  [Team](https://2017.igem.org/Team%3AUCopenhagen/Team)
  [Attributions](https://2017.igem.org/Team%3AUCopenhagen/Attributions)
  [Collaborations](https://2017.igem.org/Team%3AUCopenhagen/Collaborations)
* [Human Practices](https://2017.igem.org/Team%3AUCopenhagen/HP)

  [Overview](https://2017.igem.org/Team%3AUCopenhagen/HP/Silver)
  [Ethics](https://2017.igem.org/Team%3AUCopenhagen/HP/Ethics)
  [Integrated](https://2017.igem.org/Team%3AUCopenhagen/HP/Gold_Integrated)
  [Events](https://2017.igem.org/Team%3AUCopenhagen/HP/Events)
  [Media](https://2017.igem.org/Team%3AUCopenhagen/HP/Media)

###

# 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.

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg)

  #### Interdependency

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

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg)

  #### 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)

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg)

  #### 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

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg )

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg )

  #### Number Control

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

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg )

  #### 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

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg )

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg )

  #### 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

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg )

  #### 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

  Making first transformations in all subprojects.

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg )

  #### 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!

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg )

  #### 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

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg )

  #### Protein Import

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

### Week 31 (July 31 - August 6)

---

* #### Wet Lab Overview

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg )

  #### 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

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg )

  #### 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)

---

* #### Wet Lab Overview

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg )

  #### Number Control

  Growth curve via CFU enumeration

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg )

  #### Protein Import

  Verification of pET102 iGEM with digestion and sequencing.

### Week 33 (August 14 - 20)

---

* #### Wet Lab Overview

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg )

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg )

  #### 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)

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg )

  #### 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

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg )

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg )

  #### Number Control

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

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg )

  #### 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

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg )

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg )

  #### 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

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg )

  #### Protein Import

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

### Week 36 (September 4-10)

---

* #### Wet Lab Overview

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg)

  #### 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!

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg)

  #### 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

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg)

  #### 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)

---

* #### Wet Lab Overview

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg)

  #### Interdependency

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

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg)

  #### 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)

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg)

  #### 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)

---

* #### Wet Lab Overview

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg)

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg)

  #### Number Control

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

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg)

  #### Protein Import

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

### Week 39 (September 25 - October 1)

---

* #### Wet Lab Overview

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

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg)

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg)

  #### Protein Import

  Inoculation of YFP and BFP Biobricks

### Week 40 (October 2 - 8)

---

* #### Wet Lab Overview

  Biobricks of YFP, BFP, TrpE and AroG.

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg)

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg)

  #### 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)

---

* #### Wet Lab Overview

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg)

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg)

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg)

  #### 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)

---

* #### Wet Lab Overview

  Insertion of most Biobricks in submission vector.

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg)

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg)

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg)

  #### 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)

---

* #### Wet Lab Overview

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

  ![](https://static.igem.org/mediawiki/2017/f/fd/Interdependency_small.jpg)

  #### 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.

  ![](https://static.igem.org/mediawiki/2017/8/89/Numbercontrol_small.jpeg)

  #### Number Control

  Termination of number control sub-project.

  ![](https://static.igem.org/mediawiki/2017/a/a6/Proteinimport_small.jpg)

  #### 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)

---

* #### Wet Lab Overview

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

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# Team:UCopenhagen/Experiments

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# Team:UCopenhagen/Results

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[![](https://static.igem.org/mediawiki/2017/f/f2/UCopenhagen_logo.png)](https://2017.igem.org/Team%3AUCopenhagen)

* [Project](https://2017.igem.org/Team%3AUCopenhagen/Project)

  [Project description](https://2017.igem.org/Team%3AUCopenhagen/Project)
  [Interdependency](https://2017.igem.org/Team%3AUCopenhagen/Interdependency)
  [Number control](https://2017.igem.org/Team%3AUCopenhagen/Number-Control)
  [Protein import](https://2017.igem.org/Team%3AUCopenhagen/Protein-Import)
  [Results](https://2017.igem.org/Team%3AUCopenhagen/Results)
  [Parts](https://2017.igem.org/Team%3AUCopenhagen/Parts)
  [Modelling](https://2017.igem.org/Team%3AUCopenhagen/Model)
  [Interlab](https://2017.igem.org/Team%3AUCopenhagen/InterLab)
* [Safety & Labnotes](https://2017.igem.org/Team%3AUCopenhagen/Safety)

  [Safety](https://2017.igem.org/Team%3AUCopenhagen/Safety)
  [Notebook](https://2017.igem.org/Team%3AUCopenhagen/Notebook)
  [Protocols](https://2017.igem.org/Team%3AUCopenhagen/Protocols)
  [Improve](https://2017.igem.org/Team%3AUCopenhagen/Improve)
* [People](https://2017.igem.org/Team%3AUCopenhagen/Team)

  [Team](https://2017.igem.org/Team%3AUCopenhagen/Team)
  [Attributions](https://2017.igem.org/Team%3AUCopenhagen/Attributions)
  [Collaborations](https://2017.igem.org/Team%3AUCopenhagen/Collaborations)
* [Human Practices](https://2017.igem.org/Team%3AUCopenhagen/HP)

  [Overview](https://2017.igem.org/Team%3AUCopenhagen/HP/Silver)
  [Ethics](https://2017.igem.org/Team%3AUCopenhagen/HP/Ethics)
  [Integrated](https://2017.igem.org/Team%3AUCopenhagen/HP/Gold_Integrated)
  [Events](https://2017.igem.org/Team%3AUCopenhagen/HP/Events)
  [Media](https://2017.igem.org/Team%3AUCopenhagen/HP/Media)

###

# R E S U L T S

---

## Introduction

On this page, we will present results from the experiments we have done within our three subprojects.

The results we are the most proud of is the successful import of fluorescent proteins labeled with CPP-tag.
On the other hand, our number control experiment didn’t show much - instead check out our modelling where we modelled how it could have worked.

## Results from interdependency

**Gene expression and tryptophan production**

In interdependency, we aimed to increase tryptophan biosynthesis in  *E.coli* by overexpressing three proteins: the aromatic amino acid transporter YddG, the first protein in the shikimate pathway DAHP synthases (AroG), and the Anthranilate synthase component 1 (TrpE), responsible for anthranilate biosynthesis , precursor of L-Tryptophan (Trp). AroG and TrpE are naturally feedback inhibited, hence we modified them to be feedback resistant. This should allow higher rates of biosynthesis. (Gu et al., 2012)

*aroG* and *trpE* were successfully mutated with the point mutation and verified by sequencing the genes inserted in our expression vector (Fig. 1).

Expression of AroG and TrpE at 30 degrees in *E. coli* BL21 was verified via histidine column purification and western blot (Fig 2).

Growth curves made of the transformed cells with empty vector, single YddG, AroG, TrpE, and combination YddG-AroG and YddG-TrpE, showed that all transformants grew well in YNB media without tryptophan (Fig 3). The lowest growth rate was observed in the transformant with the an empty vector (Control in Fig 3)

![](https://2017.igem.org/File:UCopenhagen_trpE_aroG_sequencing.png)

**Figure 1**  In this figure the comparison between the natural Anthranilate synthase component 1 (TrpE) (a) and Phospho-2-dehydro-3-deoxyheptonate aldolase, Phe-sensitive (AroG) (b) and their respective negative feedback resistant (frb) version we obtained is shown. Above the DNA sequence, and the respective amino acid sequence, of the natural version is shown. Below the data obtained via the sequencing (Macrogen commercial service) of our modified genes. Is possible to see that TrpE becomes feedback resistant when 878T is substituted with cytosine, resulting in 293Met->Thr. Similarly, for AroG, the amino acid substitution 150Pro->Leu results in feedback resistance.

![](https://static.igem.org/mediawiki/2017/f/f0/AroG-trpE_Western.png)

**Figure 2** Western blot showing the expression levels of his-tagged AroG and TrpE expressed at 30℃. Three cultures of bacteria with empty expression vector, TrpE, or AroG were run. Size of AroG is 40 kDa, TrpE is 59 kDa.

![](https://static.igem.org/mediawiki/2017/e/e1/UCopenhagen_E.coli_growth_curve_transformed.png)

**Figure 3**Growth curves of *E.coli* in YNB pH 7.2 without tryptophan grown at 37 °C. *E. coli* transformed with vectors containing the different genes. Induction with 1 mM IPTG was performed 3 hours after inoculation. CK is empty vector. All measurement points are averages of two cultures.

**Tryptophan production**

The expression of the proteins and growth of the cells, however, did not result in an increased production of tryptophan.

Tryptophan concentration both in the media and from the cell lysate were measured with HPLC-MS. All concentrations measured in the media, however, were below the limit of quantification (LOQ), most being around 200 µg/L. In the cell lysate, concentrations above LOQ was only measured in two strains, and only after 58 hours growth: AroG (532.8 µg/L) and YddG-TrpE (404.2 µg/L).

A higher concentration in cell lysate indicates that tryptophan synthesis is increased, but the export is not keeping up. However, even the 0.5 mg/L detected from the cell lysate is far below the 76 mg/L tryptophan in synthetic yeast media we aimed for.

The tryptophan concentration in the media cleared from cells would be available for yeast growth in our serial growth experiments, or for the host cell in a real endosymbiotic system.

Serial growth experiments performed in YNB media with tryptophan shows unhindered yeast growth, indicating that *E. coli* and yeast should be able to co-live (Fig 4).

On the other hand, yeast in YNB without tryptophan that has to depend on tryptophan produced by our transformed *E. coli*, showed no growth, irrespective of the time *E. coli* had grown in the media (Fig 5). This finding fits with the tryptophan concentrations measured via HPLC-MS.

![]( https://static.igem.org/mediawiki/2017/2/2a/UCopenhagen_serialgrowth1.png)

**Figure 4**Growth curves of Yeast grown in media with previous E.coli growth without added tryptophan

![](https://static.igem.org/mediawiki/2017/7/74/UCopenhagen_serialgrowth2.png)

**Figure 5**Growth curve of yeast grown in YNB- media after media was cleared of our different *E. coli* strains. E.coli had grown in the media for 10.5, 31 or 58 hours before being removed by spinning and filtration to remove.

---

## Results from number control

**Sequencing og sgRNA**

![](https://2017.igem.org/File:UCopenhagen_pgRNA123_sequencing.png)

**Figure 6**Sequencing data of the plasmids pgRNA1, pgRNA2, pgRNA3 indicating that the proper seed sequence (highlighted in yellow) and sgRNA scaffold (following) has been inserted in all three plasmids.

**Growth curve of induced/uninduced**

The results obtained in number control sub-project were non-significant, therefore we decide to expand the modelling aspect of cell replication control. However, the results obtained in the wet-lab are described in the following paragraphs.

The data of the growth curve obtained via OD600 measurements without serial dilutions suggests the presence of detrimental effect on growth rate by all three construct pdCas9 – pgRNA1, pdCas9 – pgRNA2, pdCas9 – pgRNA3 when dCas9 expression is induced via tetracycline (200 ng/mL). However, further repetition of the experiment, with tuning of the protocol, did not significantly confirmed this trend.

![](https://2017.igem.org/File:UCopenhagen_growthcurvev1.png)

**Figure 7**The figure shows the effect of dCas9-sgRNA complex targeting the OriC on the bacterial growth rate. Growth was measured via OD600 and the difference in density between inoculation time and 240 min incubation at 37 °C, 220 rpm was calculated. This suggests a detrimental effect of the system of growth when dCas9 is expressed (induction using tetracycline).

---

## Results from protein Import

In protein import, our aim is to express and import a fluorescent proteins linked to a 9 Arginine cell penetrating peptide (CPP).

**Expression of CPP and fluorescent proteins**

Cells transformed with empty vector, CPP, YFP or BFP alone, or CPP-tagged fluorescent proteins (CPP-YFP, CPP-BFP) grew linearly both when induced and uninduced (Fig 8, 9 and 10). The emission of YFP and BFP were unaffected by CPP linkage. (Fig 11)

![](https://static.igem.org/mediawiki/2017/f/fd/UCopenhagen_YFP.png)

**Figure 8**Growth curve of *E. coli* transformed with CPP-YFP, YFP or empty vector (CK). Dashed lines: uninduced, solid lines induced with 1 mM IPTG.

![](https://static.igem.org/mediawiki/2017/f/fa/UCopenhagen_BFP.png)

**Figure 9**Growth curve of *E. coli* transformed with CPP-BFP, BFP or empty vector (CK). Dashed lines: uninduced, solid lines induced with 1 mM IPTG.

![](https://static.igem.org/mediawiki/2017/5/5e/UCopenhagen_CPPgrowth.png)

**Figure 10**Growth curve of *E. coli* transformed with CPP or empty vector (CK). Dashed lines: uninduced, solid lines induced with 1 mM IPTG.

![](https://static.igem.org/mediawiki/2017/a/a2/CPP-Export-E.coli.png)

**Figure 11**Normal and fluorescence microscopy of *E. coli* cells expressing (A-C): Empty vector, (D-F) CPP, (G) BFP, (H) YFP, (I) CPP-BFP, (J) CPP-YFP. Visualized with (A+D) Normal settings, (B, E, G and I) BFP filter settings, and (C,H and I) YFP filter settings. YFP filter settings: excitation 510-555 nm and emission 510-555 nm. BFP filter settings: excitation 330-385 and emission >420 nm.

**Purification**

The fluorescent proteins with and without CPP tags were purified before testing the uptake into different *E. coli* cells via SDS-page and immunoblotting (His-tag). (Fig 12 and 13)

![](https://static.igem.org/mediawiki/2017/2/29/SDS_page_CK-CPP-CPP-BFP.png)

**Figure 12**SDS page of proteins purified from E coli cells with empty vector (CK), CPP alone and CPP-BFP. Numbers above the lanes is the elution number. Elution number 1 (combining the samples loaded on the two lanes within the red boxes) was used to test import.

![](https://static.igem.org/mediawiki/2017/a/a3/SDS_page_BFP-YFP-CPP-YFP.png)

**Figure 13** SDS page of proteins purified from E coli cells with BFP, YFP and CPP-YFP. Numbers above the lanes is the elution number. Elution number 1 (combining the samples loaded on the two lanes within the red boxes) was used to test import.

**Import**
Functionality of CPP in facilitating the fluorescent proteins cellular internalization was tested using fluorescent microscopy. Purified proteins CPP, YFP, BFP, CPP-YFP, or CPP-BFP were added to *E. coli* cultures. These were incubated for 10 minutes, washed to remove extracellular (or non membrane-bounded) proteins. Finally visualisation via fluorescence microscopy (Olymus BX60) was carried out. Fig 14 shows that treated cells emit fluoresc after treatment with CPP tagged YFP/BFP, but not when treated with untagged fluorescent proteins. This is a strong indicator that the CPP tag facilitates import of proteins in *E. coli*.

![](https://static.igem.org/mediawiki/2017/c/c7/CPP_import_E.coli_fluorescens.png)

**Figure 14**Fluorescence microscopy images of import experiment in *E.coli*. (A+B) BFP, (C+D)CPP-BFP, (E+F)YFP, (G+H)CPP-YFP. Top row is normal settings, while (B+D) is BFP filter settings and (F+H) is YFP filter settings. YFP filter settings: excitation 510-555 nm and emission 510-555 nm. BFP filter settings: excitation 330-385 and emission >420 nm. Images are cropped to focus on cells expressing fluorescence when any are observed.

---

## Conclusions

**In Interdependency,** we had successfully transformed and expressed three genes involved in tryptophan production and export. Namely, AroG, TrpE, and YddG. However, the level of tryptophan produced by our *E. coli* strains is not sufficient to sustain the growth of tryptophan auxotroph yeast. The level of intracellular Trp in *E. coli* showed an increase in the strains AroG and YddG-TrpE. Moreover, our experiment show that *E. coli* is able to grow onto yeast YNB media. This gave us a reason to say that a relationship between the two cells is not impossible.

**In Number Control,** we successfully created three sgRNA and separately transformed cells with these and a dCas9 expression plasmid. However, induction of dCas9 did not lead to a significant inhibition of growth as expected. Therefore, we continue our investigation of the number control mechanism in our modelling.

**In Protein impor,** we expressed fluorescent proteins with and without a cell penetrating peptide (CPP). This showed that the fluorescent proteins emission is not affected by the 9 arginine tag. And that CPP greatly enhanced the colocalization of cell and fluorescent CPP tagged proteins. Preliminary results (shown on the registry page of our cell penetrating USER casette biobrick) suggests that this mechanism is present also in other bacterial taxa (*Pseudomona aeruginosa*).

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* [Introduction](#Top)
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