iGEM 2018 — Team Aalto-Helsinki

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# Team:Aalto-Helsinki/Notebook

[![](https://static.igem.org/mediawiki/2018/thumb/4/41/T--Aalto-Helsinki--Silkolor.png/1198px-T--Aalto-Helsinki--Silkolor.png)](https://2018.igem.org/Team%3AAalto-Helsinki)

* Project

  [Project Description](https://2018.igem.org/Team%3AAalto-Helsinki/Description)
  [Product Design](https://2018.igem.org/Team%3AAalto-Helsinki/Applied_Design)
* Wet Lab

  [Background](https://2018.igem.org/Team%3AAalto-Helsinki/Background)
  [Experiments](https://2018.igem.org/Team%3AAalto-Helsinki/Experiments)
  [Results](https://2018.igem.org/Team%3AAalto-Helsinki/Demonstrate)
  [InterLab](https://2018.igem.org/Team%3AAalto-Helsinki/InterLab)
* Dry Lab

  [Model](https://2018.igem.org/Team%3AAalto-Helsinki/Model)
* Parts

  [Basic Part](https://2018.igem.org/Team%3AAalto-Helsinki/Basic_Part)
  [Composite Part](https://2018.igem.org/Team%3AAalto-Helsinki/Composite_Part)
* Human Practices

  [Integrated Human Practices](https://2018.igem.org/Team%3AAalto-Helsinki/Human_Practices)
  [Education & Public Engagement](https://2018.igem.org/Team%3AAalto-Helsinki/Public_Engagement)
  [Collaborations](https://2018.igem.org/Team%3AAalto-Helsinki/Collaborations)
* Team

  [Team Members](https://2018.igem.org/Team%3AAalto-Helsinki/Team)
  [Sponsors](https://2018.igem.org/Team%3AAalto-Helsinki/Sponsors)
  [Attributions](https://2018.igem.org/Team%3AAalto-Helsinki/Attributions)
* Achievements

  [Achievements](https://2018.igem.org/Team%3AAalto-Helsinki/Achievements)
  [Judging Form](https://igem.org/2018_Judging_Form?team=Aalto-Helsinki)

# Notebook

### Day 1

Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Proin sed libero enim sed faucibus turpis in eu mi. Cras adipiscing enim eu turpis egestas pretium aenean pharetra. Velit egestas dui id ornare arcu odio ut sem nulla. Nullam non nisi est sit amet facilisis. Maecenas ultricies mi eget mauris pharetra et.

Quis ipsum suspendisse ultrices gravida dictum. Porttitor rhoncus dolor purus non enim praesent elementum. Amet mauris commodo quis imperdiet massa. Fames ac turpis egestas maecenas pharetra convallis posuere morbi leo. Eget sit amet tellus cras adipiscing enim eu turpis. Sem integer vitae justo eget magna fermentum iaculis. Quam elementum pulvinar etiam non quam lacus. Feugiat pretium nibh ipsum consequat nisl vel pretium. Ullamcorper morbi tincidunt ornare massa eget.

Neque vitae tempus quam pellentesque nec nam aliquam sem. Tellus rutrum tellus pellentesque eu tincidunt tortor aliquam nulla. Diam volutpat commodo sed egestas egestas fringilla. Congue eu consequat ac felis donec et odio pellentesque diam. Egestas sed tempus urna et pharetra pharetra massa. Mauris sit amet massa vitae tortor condimentum. Diam quis enim lobortis scelerisque fermentum dui faucibus in ornare.

Condimentum mattis pellentesque id nibh tortor id. Duis ultricies lacus sed turpis tincidunt id aliquet risus feugiat. Mi quis hendrerit dolor magna eget est. Nec tincidunt praesent semper feugiat. Varius morbi enim nunc faucibus a pellentesque sit amet. Adipiscing elit duis tristique sollicitudin nibh sit amet. Elit ullamcorper dignissim cras tincidunt lobortis feugiat vivamus.

### Day 2

Gravida dictum fusce ut placerat. Leo vel fringilla est ullamcorper eget nulla facilisi etiam. Cursus in hac habitasse platea dictumst quisque sagittis. Eu volutpat odio facilisis mauris. Feugiat vivamus at augue eget arcu. Netus et malesuada fames ac turpis egestas maecenas pharetra. Purus in massa tempor nec. Dolor sit amet consectetur adipiscing elit. Nunc sed augue lacus viverra vitae congue eu. Ut pharetra sit amet aliquam. Venenatis lectus magna fringilla urna porttitor rhoncus. Nunc non blandit massa enim nec dui nunc mattis enim. Posuere sollicitudin aliquam ultrices sagittis orci a scelerisque. Imperdiet proin fermentum leo vel. Vivamus arcu felis bibendum ut tristique et egestas quis. Sed risus ultricies tristique nulla aliquet enim tortor. Id porta nibh venenatis cras sed felis eget velit aliquet. Ac turpis egestas integer eget aliquet nibh praesent tristique magna. Arcu dictum varius duis at consectetur lorem donec massa.

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[![](https://static.igem.org/mediawiki/2018/0/06/T--Aalto-Helsinki--Aalto.png)](http://www.aalto.fi/en/)
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# Team:Aalto-Helsinki/Experiments

[![](https://static.igem.org/mediawiki/2018/thumb/4/41/T--Aalto-Helsinki--Silkolor.png/1198px-T--Aalto-Helsinki--Silkolor.png)](https://2018.igem.org/Team%3AAalto-Helsinki)

* Project

  [Project Description](https://2018.igem.org/Team%3AAalto-Helsinki/Description)
  [Product Design](https://2018.igem.org/Team%3AAalto-Helsinki/Applied_Design)
* Wet Lab

  [Background](https://2018.igem.org/Team%3AAalto-Helsinki/Background)
  [Experiments](https://2018.igem.org/Team%3AAalto-Helsinki/Experiments)
  [Results](https://2018.igem.org/Team%3AAalto-Helsinki/Demonstrate)
  [InterLab](https://2018.igem.org/Team%3AAalto-Helsinki/InterLab)
* Dry Lab

  [Model](https://2018.igem.org/Team%3AAalto-Helsinki/Model)
* Parts

  [Basic Part](https://2018.igem.org/Team%3AAalto-Helsinki/Basic_Part)
  [Composite Part](https://2018.igem.org/Team%3AAalto-Helsinki/Composite_Part)
* Human Practices

  [Integrated Human Practices](https://2018.igem.org/Team%3AAalto-Helsinki/Human_Practices)
  [Education & Public Engagement](https://2018.igem.org/Team%3AAalto-Helsinki/Public_Engagement)
  [Collaborations](https://2018.igem.org/Team%3AAalto-Helsinki/Collaborations)
* Team

  [Team Members](https://2018.igem.org/Team%3AAalto-Helsinki/Team)
  [Sponsors](https://2018.igem.org/Team%3AAalto-Helsinki/Sponsors)
  [Attributions](https://2018.igem.org/Team%3AAalto-Helsinki/Attributions)
* Achievements

  [Achievements](https://2018.igem.org/Team%3AAalto-Helsinki/Achievements)
  [Judging Form](https://igem.org/2018_Judging_Form?team=Aalto-Helsinki)

# Experiments

- Table of Contents
[Abstract](#collapseExample16)- Experiments
[Cloning](#collapseExample1)
Protein Production and Purification
Testing- Protocols
Silk Growing and Purification
Preparation of an Agarose Gel (1%) & Electrophoresis
Preparation of an SDS-PAGE Gel and Electrophoresis
Preparation of Competent *E. coli* Cells
Transforming Chemically Competent *E. coli* Cells Using Heat Shock
Golden Gate Assembly
Plasmid Isolation
Digestion
Ligation
Protein Purification with ÄKTA
Concentration of Proteins Using Sartorius Vivaspin 20 Ultracentrifugation Tubes
Microtitre Plate Growth Experiment- Other
Sequences

Abstract

The parts of our fusion proteins were ordered as separate sequences: mRPF, Prancer Purple, sgBP, silk repeats, cellulose binding module, and keratin-binding domain. Constructs containing a chromoprotein sequence, a binding domain, and silk repeats in different combinations were assembled with Golden Gate. Purified and evaluated plasmids were transformed in *E. coli* production strain BL21, and the constructs were produced by inducing with IPTG. Proteins were purified with ÄKTA or a heat-based protocol based on our modeling results.

The tests performed included:

* The effect of protein production on cell growth.
* The binding of the cellulose binding module to nanocellulose and untreated cotton.
* The binding of the keratin binding domain to a keratin supplement and hair.
* The production of colorful silk fibers.

Sequences

The following sequences were ordered as gBlocks from IDT, except ADF3 which was ordered from GeneArt. All the sequences were designed with BsaI sites and overhangs that bind with the correct sequence that the part is linked to. Constructs can be assembled and ligated to the plasmid at the same time with Golden Gate assembly. BsaI sites are underlined.

mRFP + N-terminal linker:

GGTCTCCATGGGCATGGCGTCATCAGAGGATGTCATAAAAGAATTTATGC
GGTTTAAAGTAAGAATGGAAGGTTCGGTCAACGGACATGAGTTTGAGATT
GAAGGCGAAGGGGAAGGTAGACCATACGAAGGTACACAGACAGCTAAGCT
TAAGGTTACTAAAGGAGGCCCCTTGCCGTTCGCTTGGGATATTCTTTCCC
CACAATTCCAGTACGGTAGCAAAGCCTATGTTAAACACCCAGCCGACATT
CCAGACTATCTGAAGCTTAGCTTTCCCGAAGGATTCAAGTGGGAACGGGT
CATGAATTTTGAAGATGGGGGGGTGGTAACTGTTACGCAGGACTCTTCGT
TGCAGGATGGTGAATTTATCTATAAAGTCAAGCTTCGCGGAACTAACTTT
CCGTCGGATGGACCCGTCATGCAAAAGAAAACCATGGGATGGGAGGCCAG
CACGGAACGTATGTATCCCGAGGACGGTGCCTTGAAAGGTGAAATAAAAA
TGAGATTGAAATTAAAGGACGGTGGACACTACGATGCCGAAGTCAAAACA
ACATACATGGCTAAGAAGCCGGTTCAATTGCCGGGCGCATACAAGACCGA
CATAAAATTGGATATCACGTCACACAACGAGGATTACACTATCGTAGAGC
AGTACGAACGCGCCGAGGGGCGGCACTCCACCGGAGCTTCAGCTAGCGCC
AGTGCCAGTGCCGGTGCGTCGGCAGCAGCTTCAGCAGGAGCAAGCGAGAG
ACC

PrancerPurple + N -terminal linker:

GGTCTCCATGGGCATGGCGTCTCTGGTTAAAAAAGACATGTGCATCAAAA
TGACCATGGAAGGTACCGTTAACGGTTACCACTTCAAATGCGTTGGTGAA
GGTGAAGGTAAACCGTTCGAAGGTACCCAGAACATGCGTACCCGTGTTAC
CGAAGGTGGTCCGCTGCCGTTCGCGTTCGACATCCTGGCGCCGTGCTGCA
TGTACGGTTCTAAAACCTTCATCAAACACGTTTCTGGTATCCCGGACTAC
TTCAAAGAATCTTTCCCGGAAGGTTTCACCTGGGAACGTACCCAGATCTT
CGAAGACGGTGGTGTTCTGACCGCGCACCAGGACACCTCTCTGGAAGGTA
ACTGCCTGATCTACAAAGTTAAAGTTCTGGGTACCAACTTCCCGGCGAAC
GGTCCGGTTATGCAGAAAAAAACCGCGGGTTGGGAACCGTGCGTTGAAAT
GCTGTACCCGCGTGACGGTGTTCTGTGCGGTCAGTCTCTGATGGCGCTGA
AATGCACCGACGGTAACCACCTGACCTCTCACCTGCGTACCACCTACCGT
TCTCGTAAACCGTCTAACGCGGTTAACATGCCGGAATTTCACTTCGGTGA
CCACCGTATCGAAATCCTGAAAGCGGAACAGGGTAAATTCTACGAACAGT
ACGAATCTGCGGTTGCGCGTTACTCTGACGTTCCGGAAAAAGCGACCTCT
GCGTCTGCGTCTGCGTCTGCGGGTGCGTCTGCGGCGGCGTCTGCGGGTGC
GAGCGAGAGACC

ADF-3, 6 repeats with BsaI sites:

GGTCTCAAGCGCGGGGGCTGGCCCCTAC GGACCAGGAGCCTCTGCGGCGGCGGCGGCAGCAGGGGGTTATGGTCCCGG
AAGCGGGCAACAGGGTCCGGGTCAACAAGGACCCGGACAACAAGGACCGG
GACAGCAGGGGCCTGGCCAGCAGGGACCGTACGGACCAGGTGCATCTGCG
GCAGCGGCGGCGGCGGGCGGATATGGACCAGGTTCAGGTCAACAGGGACC
AGGCCAACAGGGGCCTGGGCAACAAGGCCCGGGTCAACAGGGCCCTGGAC
AACAGGGTCCGTATGGTCCCGGCGCGTCCGCGGCAGCGGCGGCGGCCGGG
GGGTACGGTCCGGGGTCCGGGCAACAGGGTCCTGGCCAACAAGGCCCAGG
GCAGCAGGGCCCTGGACAGCAAGGACCTGGACAGCAAGGTCCGTACGGGC
CAGGTGCTTCTGCGGCAGCGGCGGCGGCAGGGGGATACGGGCCGGGCTCA
GGGCAGCAGGGGCCAGGCCAGCAGGGCCCTGGCCAACAAGGGCCGGGGCA
ACAGGGACCTGGCCAGCAAGGACCGTACGGCCCTGGCGCTAGTGCCGCTG
CAACCGCTGCCGGGGGTTACGGCCCGGGTTCCGGCCAACAAGGCCCCGGA
CAGCAAGGACCCGGTCAACAGGGACCCGGACAGCAGGGACCAGGGCAACA
AGGACCTTATGGACCAGGGGCTTCTGCCGCAGCAGCAGCCGCAGGGGGCT
ACGGCCCAGGGAGCGGTCAACAGGGACCCGGCCAGCAGGGCCCAGGGCAG
CAGGGTCCCGGACAGCAAGGGCCAGGACAACAAGGTTCTAAGAGACC

CBM3:

GGTCTCAAGCGCGATGGGTAACTTAAAGGTTGAGTTCTATAACAGTAATC
CGTCTGACACAACGAATAGCATTAATCCTCAGTTCAAGGTGACAAACACT
GGTTCTAGTGCTATTGATCTGAGTAAGCTGACACTTAGATATTACTATAC
GGTCGATGGTCAAAAGGATCAAACTTTTTGGTGCGATCACGCCGCGATAA
TCGGTAGTAACGGATCGTATAACGGGATTACGTCCAATGTGAAAGGCACA
TTCGTCAAAATGTCCTCTTCCACGAATAACGCCGATACATACTTGGAGAT
AAGTTTCACCGGCGGCACTTTGGAGCCCGGAGCGCATGTTCAAATCCAGG
GAAGATTCGCCAAAAACGATTGGAGCAATTATACGCAGAGTAATGACTAC
TCCTTCAAATCTGCATCTCAGTTTGTTGAGTGGGATCAAGTTACCGCTTA
CCTTAACGGTGTTTTAGTATGGGGAAAAGAACCACTCGAGAGAGACC

KBD:

GGTCTCAAGCGCGATGTTCAGCGACACTTTAGAAGAATCTTCCCCAATTG
CCGCCATATTCGACACGGAAAACCTTGAGAAAATCTCTATTACGGAGGGG
ATAGAGAGAGGCATAGTTGATTCGATAACCGGGCAGCGGTTACTTGAAGC
ACAGGCTTGTACCGGAGGGATCATTCATCCGACTACTGGGCAGAAGTTGT
CTTTACAGGACGCTGTCAGCCAAGGGGTAATAGATCAGGACATGGCGACC
CGTCTGAAGCCAGCTCAGAAGGCGTTTATCGGTTTCGAAGGGGTAAAAGG
AAAAAAAAAAATGTCTGCCGCTGAAGCAGTCAAAGAAAAATGGCTTCCCT
ACGAGGCAGGACAGCGCTTCCTTGAATTTCAGTATCTGACTGGGGGGCTG
GTGGACCCAGAAGTCCACGGGCGTATCTCCACGGAAGAGGCCATACGCAA
GGGATTTATAGACGGAAGAGCAGCTCAGCGGCTGCAAGATACATCCTCTT
ACGCGAAAATTTTGACGTGTCCTAAAACGAAGCTGAAAATCTCTTATAAG
GACGCTATAAATCGCTCCATGGTGGAAGATATTACGGGCTTACGCCTGCT
GGAGGCGGCTTCAGTATCGTCTAAAGGCTTGCCGAGCCCCTACAATATGT
CTTCAGCCCTCGAGAGAGACC

CBM and KBD were also ordered with a C-terminal linker at the beginning of the sequence so that they could be assembled with ADF3.

Linkers used:

C-terminal linker ASASAAASAASTVANS

N-terminal linker SASASASAGASAAASAGA

Cloning

We ordered each part of our fusion proteins as a separate sequence: mRPF, Prancer Purple, sgBP, silk repeats, cellulose binding module, and keratin-binding domain. Some of the proteins were designed so that they had a 23-amino-acid linker to facilitate the folding and function of the individual proteins within the fusion protein. [Our DNA sequences](#collapseExample17) were synthesised by IDT, except for the silk repeats which were produced by GeneArt.

To assemble the sequences, we used [Golden Gate assembly](#collapseExample9). The Golden Gate assembly enables the assembly of multiple sequences by using only one enzyme, BsaI. The sequences were designed so that they had the BsaI recognition site at both sides. In this way, after the digestion with the enzyme, each sequence is flanked by 4-base-pair overhangs that can be used for a subsequent ligation with the desired sequence.

In each Golden Gate reaction, we joined one or more of our protein sequences with the pSVA1 plasmid. This vector was a convenient choice for Golden Gate assembly, as it is a modified version of pET28(+), having two BsaI restriction sites. Other features of the plasmid are a His6x-tag, enabling promoter protein purification, a T7lac promoter in the backbone, enabling promoter induction with IPTG, and kanamycin resistance.

![](https://static.igem.org/mediawiki/2018/9/9a/T--Aalto-Helsinki--plasmidd.png)

Figure 1. Plasmid map of pSVA1 (Geneious version 11.1.5).

By using Golden Gate, we assembled the following constructs:

>mRFP - N linker - CBM

>mRFP - N linker - KBD

>prancerPurple - N linker - CBM

>prancerPurple - N linker - KBD

>mRFP - N linker - silk

>prancerPurple - Nlinker - silk

>mRFP - N linker - silk - CBM

>mRFP - N linker - silk - KBD

>prancerPurple - N linker - silk - CBM

>prancerPurple - N linker - silk - KBD

We also assembled constructs containing the chromoprotein sgBP, but for those, the cloning was unsuccessful.

The plasmids, assembled with Golden Gate, were then transformed in DH10B *Escherichia coli* strain, which is optimal for cloning. The cells were made competent with the [competent cells protocol.](#collapseExample7)

The transformant colonies were cultivated and the plasmids were purified from them with the Macherey-Nagel Nucleospin Plasmid kit. In order to check whether the vectors had the correct insert, they were [digested](#collapseExample11) with the enzymes NcoI and XhoI. The fragment sizes were then evaluated with a [1% agarose gel run.](#collapseExample5)

Protein Production and Purification

After ensuring that we had the right constructs, we purified the plasmids again using the Nucleospin Plasmid kit and transformed them in *E. coli* strain BL21. This was our production strain, as it enabled the expression of the fusion proteins, regulated by the T7lac promoter, by adding IPTG. We produced the constructs by inducing with a final concentration of 0,5 mM IPTG.

![](https://static.igem.org/mediawiki/2018/b/bb/T--Aalto-Helsinki--flasks.png)

Figure 2. The induced cultures, expressing our colorful fusion proteins.

The proteins were then purified with two different methods. The proteins not containing silk were [purified with ÄKTA](#collapseExample13). However, this method is time-consuming and not suitable for purifying large amounts of proteins. Since the silk repeats have an unfolded structure, silk proteins can be purified with a protocol that involves heat to lyse the cells. Prancer Purple chromoprotein and the cellulose binding module we used are thermostable, so the [silk purification protocol](#collapseExample4) could be used to purify the constructs containing them. To find out whether the treatment would affect the stability of mRFP and keratin-binding domain, we modeled their behavior within [the range of temperatures](https://2018.igem.org/Team%3AAalto-Helsinki/Model) we would use. As the modeling results suggested that the proteins did not encounter any significant changes in structure within the temperature range, we purified all the silk-containing fusion proteins with the heat-based protocol.

![](https://static.igem.org/mediawiki/2018/c/c4/T--Aalto-Helsinki--tubes.png)

Figure 3. Our red and purple purified proteins.

Testing

**Cytation**

Since our fusion proteins are quite big, we wanted to test how the protein production affects the cell growth. *E. coli* strain BL21 with mRFP, mRFP-CBM, mRFP-KBD, or mRFP-silk producing plasmid were induced with 0.1, 0.5, or 1mM IPTG. We also had controls of each which were not induced.

**Cellulose binding**

The cellulose binding module was tested with [SDS-page](#collapseExample6). As cellulose, we used nanofibrils (CNF) derived from birch. 1%(w/v) nanocellulose solutions were prepared from stock concentration, and a binding test was conducted with different concentrations of proteins to CNF (1:1, 1:10 and 1:100).

We also used our proteins containing the cellulose binding module to dye pure, untreated cotton. The test was performed by leaving a piece of cotton shaking in a dye bath covered with protein solution overnight. Color fastness was tested by washing the cotton with lukewarm water in shaking for 1 hour.

**Keratin binding**

We did not have the time or the resources to purchase pure keratin products for the test, so we tested KBD binding to keratin with Evonia Keratin+Biotin supplement. The supplement contains about 95% of keratin powder which we dissolved to water, and did a similar binding test as with cellulose.

One of our interests was to try if KBD would bind to hair keratin. We had a 1g sample of light blond hair which we covered with concentrated PSK and incubated in room temperature for 2 hours.

**Silk fibers**

We followed a protein concentration protocol in order to obtain colorful silk fibers.
The protein was concentrated with Econo-Pac® 10DG Desalting Prepacked Gravity Flow Columns. In principle, when the silk protein is concentrated, silk repeats interact with each other, forming a fiber. We tried to make fibers by inserting tweezers in our concentrated solution and carefully opening them. We added a drying step by using hot air to further concentrate the solution.

Proteins were concentrated using the [Sartorius Vivaspin 20 ultrafiltration centrifugal columns 30k MWCO](#collapseExample14) protocol.

![](https://static.igem.org/mediawiki/2018/a/a7/T--Aalto-Helsinki--concentration.png)

Figure 4. To concentrate our fusion proteins, we used Econo-Pac columns.

Silk Growing and Purification

**Day 1**

* Inoculate 1 colony to a 50 mL preculture of LB with 50 μg/mL kanamycin
* Let grow ~7 hours at +37 °C, 220 rpm
* Check OD, if over 0.8, dilute with LB + 50 μg/mL kanamycin
* Inoculate entire 50 mL culture into 450 mL of LB + 50 μg/mL kanamycin into a 1-2 L flask covered with air-permeable membrane (using a large enough flask is important so the culture gets enough oxygen).
* Induce with IPTG (final concentration 0.5mM)
* Grow overnight at +30 °C 160 rpm

+ The cells should have at least 18 hours to grow to maximize protein yield.

**Day 2**

* Collect the cells by centrifuging 30 minutes at 5 000 - 10 000 g (if possible, use 10 000 g)
* Use 10mL of lysing mix for 1g of cell pellet
* Lysing mix:

+ DNAse1
+ Lysozyme
+ 5mM magnesium chloride
+ 50mM Tris-HCL pH 7.4
+ Protease inhibitor cocktail tablet (1 for 100 ml)

* Resuspend and incubate at room temperature for 30 min

+ If the solution is too viscous after the incubation, add more lysozyme and DNAse

* Shearing using a sonificator

+ During sonification, keep the falcon tube where your cells are in a beaker filled with ice to prevent overheating.
+ Use a 1/16 tip with 30 % (or maximum if lower) amplitude
+ Sonificate for 5 min with pulses on and off 5 seconds at a time
+ Keep tip ~0.5 cm below surface, put lower if foaming occurs.

* Incubate in +70 °C for 30 min (water bath recommended)
* Centrifuge 5 000 - 10 000 g (preferably 10 000) for 90 min
* Collect supernatant
* Desalting (and buffer exchange) using Econopack columns

+ Prepare columns according to the manual
+ Add 3 ml of sample
+ Allow to drip into waste tube until dripping stops
+ Add 4 ml of buffer you want your proteins in (eg. MilliQ-water or 50 mM Tris-HCL)
+ Allow to drip into collection tube until dripping stops
+ Repeat steps b and c for entire sample
+ Wash column 3 x with MilliQ-water and 1 x with 20 % EtOH
+ Store column at room temperature in 20 % EtOH

Preparation of an Agarose Gel (1%) & Electrophoresis

For 2 gels:

* 1.25g of agarose
* 125mL of 1X TAE buffer
* 12.5μL of SYBR Safe DNA Gel Stain (ThermoFisher Scientific, no. S33102).

Steps:

* Weigh agarose in an Erlenmeyer flask.
* Add 1X TAE buffer and heat up the mixture in the microwave oven so that the agarose has completely dissolved.
* Cool down the solution and add 2 drops of SYBR Safe DNA Gel Stain (ThermoFisher Scientific, no. S33102).
* Pour the liquid into a casting tray, place a comb (or two combs) in the tray and let the gel solidify.
* Once solidified, place the gel in a gel electrophoresis box, remove the combs and cover the gel with 1X TAE buffer.
* Load a DNA ladder (used: ThermoFisher Scientific GeneRuler DNA Ladder Mix, ready-to-use, no. SM0333) and the samples (see: sample preparation) into the wells.
* Connect the electrodes to a power supply (negative electrode is the end where the samples are loaded and positive electrode is the other end of the gel towards which the samples run).
* Run the gel electrophoresis at 110V.

Sample preparation:

* Add 1μL of 6X DNA Loading Dye (used: ThermoFisher Scientific DNA Gel Loading Dye (6X), no. R0611) in 5μL of DNA sample.

Preparation of an SDS-PAGE Gel and Electrophoresis

For two gels:

* Build the equipment: place two glass plates against each other in a gel caster.
* Prepare a mixture for the separating gel (here 7.5%):

+ 4X separating buffer (1.5M Tris-HCl, 0.4% SDS, pH 8.8) - 3.5mL
+ H2O - 7.875 mL
+ 40% Acrylamide - 2.625 mL
+ 10% APS - 90 μL
+ TEMED - 17.5 μL
+ NOTE: Add APS and TEMED at the end, then mix the solution and pipet it between the glass
  plates. Add isopropanol on top of the gel to remove bubbles and make the surface even.
+ Let the separating gel solidify (~45min)

* Prepare a mixture for the stacking gel (here 3%):

+ 2X stacking buffer (0.5M Tris, 0.2% SDS, pH 6.8) - 3.4mL
+ H2O - 3.2 mL
+ 40% Acrylamide - 0.6 mL
+ 10% APS - 60 μL
+ TEMED - 12.5 μL
+ After the separating gel has polymerized, remove isopropanol. Pipet the stacking gel mixture on top of it and place a comb between the glass plates. Let the stacking gel polymerize.

* Place the polymerized gel(s) in a running tank and fill up the space between the gels with the 1x SDS-PAGE running buffer containing diluted from 10X SDS-PAGE stock solution of 25 mM Tris, 192 mM glycine, 0.1% SDS. Add a sufficient amount of buffer in the tank surrounding the gels.
* Remove the comb(s) and pipet a ladder (used: ThermoFisher Scientific PageRuler Prestained Protein Ladder, 10 to 180 kDa, Catalog number: 26616) and the samples (NOTE: sample processing) into the wells.
* Run the electrophoresis at 110 V for ~1-1.5 hours until the samples have run almost to the bottom of the gel.* Rinse the gels with H2O and stain them with Coomassie blue for 30-45 minutes with shaking.
  * Keep the gels in the destaining solution (30%methanol, 10%acetic acid, and water) for ~1 hour (or more) until the bands become visible and most of the blue background staining is lost. The destaining solution may be changed during this time.
  * Image the gels.

Sample processing for SDS-PAGE:

* After adding SDS Loading Dye into the samples, heat them at +95°C for 10 minutes.

Preparation of Competent *E. coli* Cells

Protocol based on CHEM-E3160, Biolab III -Laboratory Course manual, written by Heli Viskari, Mari Piirainen and Bart Rooijakkers

**All steps from harvesting the cells should be carried out at 4°C and the cells should then
be kept on ice whenever possible! Make sure the CaCl2, CaCl2+25% glycerol and Eppendorf
tubes are pre-chilled as well.**

Materials:

* *E. coli* cells (from a plate or -80°C competent stock) – Top10, XL-blue, DH5α
* LB medium (sterile)
* 0.1 M CaCl2, ice cold (sterile)
* Optional: 0.1 M CaCl2+20% glycerol, ice cold (sterile)
* 1.5 mL Eppendorf tubes, ice cold (sterile)

Steps:

* (Optional) Inoculate strain of *E. coli* into 3-5 mL LB and grow O/N at 37°C + shaking.
* Next morning, use 1 v/v % (40 μl) AND 2 v/v % (80 μl) of the O/N culture to make a 4 ml
  liquid culture of cells (LB, in a sterile plastic tube). Grow the cells at 37°C + shaking
  (~220 rpm) until the OD600 is between 0.3 – 0.4 (usually takes around 1,5 - 2 hours). Do
  NOT let the OD600 get > 0,5!!
* Keep the tubes on ice for 5-10 min.
* Divide the culture in pre-chilled Eppendorf tubes.
* Centrifuge the cells for 3 min at ~3300 g with a tabletop centrifuge. Check that there is
  a visible cell pellet on the bottom of the tube.
* Gently discard the supernatant. Place the tubes back on ice and gently resuspend the
  cells with 1 ml of cold 0,1M CaCl2 by pipetting.
* Centrifuge the cells again for 3 min at ~3300 g with a tabletop centrifuge. Check that
  there is a visible cell pellet on the bottom of the tube.
* Gently discard supernatant and resuspend each cell pellet in 100 μl of 0.1M CaCl2 (Or
  0.1M CaCl2 + 25% glycerol if the cells are frozen).
* Keep the cells on ice for 20 mins, after which they are ready for transformation.
* If needed, store possible extra tubes in -80°C freezer according to teachers' instructions.

Transforming Chemically Competent *E. coli* Cells Using Heat Shock

Protocol based on CHEM-E3160, Biolab III -Laboratory Course manual, written by Heli Viskari, Mari Piirainen and Bart Rooijakkers.

* Pipet 50 μL of competent cells and 1-8 μL of DNA (up to 100 ng) into an
  eppendorf tube, mix gently
* Incubate tubes on ice for 10-20 minutes
* Incubate tubes in 42°C for 45 sec - 1 minute = heat shock
* Incubate tubes on ice for 5 minutes
* Add 500 μL of LB medium
* Incubate cells at 37°C and 200 rpm for 30 min - 1 hour
* Collect cells by centrifugation (3 mins at 3300 g) and remove about 450 μL supernatant
* Resuspend cells in the remaining supernatant and plate onto an appropriate antibiotic
  LB-agar plate
* Incubate overnight at 37°C or over weekend in RT

Golden Gate Assembly

Golden Gate assembly mixture, working volume 15 μl:

|  |  |
| --- | --- |
| pSVA1 plasmid | 100 ng |
| Other inserts, each | 100 ng |
| BsaI | 1.5 μl |
| T4 DNA Ligase | 0.5 μl |
| T4 DNA Ligase Buffer | 2.5 μl |
| H2O | to 15 μl |

Steps:

* Add each part accordingly to sterile PCR tubes.
* Mix it properly by pipetting the solution up and down.
* Incubate in thermocycler with following program:

+ (5 min 37°C → 5 min 16°C) x 30 cycles
+ 10 min 60°C
+ (Infinite 4°C until you pick up the tubes)

Plasmid Isolation

Plasmid isolation was done using the Macherey-Nagel Nucleospin Plasmid kit. The protocol used was the “5.1 Isolation of high-copy plasmid DNA from *E. coli*”, which can be found from page 15-16 of [the kit manual.](http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NS.pdf)

Digestion

Based on [digestion protocol by Thermofischer.](https://tools.thermofisher.com/content/sfs/manuals/MAN0012413_Fast_Digestion_DNA_UG.pdf)

|  | Plasmid DNA Unpurified PCR product | |
| --- | --- | --- |
| Nuclease-free water | Fill to total volume | Fill to total volume |
| 10X FastDigest buffer | 2μL | 2μL |
| DNA\* | 2μL (up to 1μg) | 10μL (~0.2μg) |
| FastDigest enzyme | 1μL | 1μL |
| FastDigest enzyme | 1μL | 1μL |
| Total | 20μL | 30μL |

Steps:

* Prepare the reaction mixture at RT in the order indicated above
* Mix gently and spin down
* Incubate at 37°C in a heat block ~30 min
* (optional) Inactivate the enzyme for 20 minutes at the temperature sufficient for inactivating the enzymes you are using

\*Volume of the DNA can be scaled up to 10 μL or down to 0.5μL depending on the DNA concentration.

Ligation

* 10X T4 DNA ligase buffer - 2 μL
* T4 DNA ligase - 1 μL
* Vector DNA - e.g. 50ng
* Insert DNA - according to calculations
* Nuclease-free water - fill up to 20μL

Steps:

* Choose the amount of vector to be used (e.g. 50ng) and the molar ratio of the insert and the vector (we used 3:1 or 5:1).
* Calculate the amount of insert that is needed with the following formula: Insert (ng) = vector (ng) \* insert (bp)vector (bp) x 5 (here: 5:1 molar ratio).
* Prepare the ligation mixture with the restricted vector & insert, and a negative control mixture with the restricted vector only.
* Incubate the mixture at 16 °C overnight or 2 hours at room temperature.

Protein Purification with ÄKTA

* Change lanes A1 and B1 into filtered (degassed) Milli-Q. Wash the pumps and system with MQ. Then wash the injection loop with 10 x column volume of MQ. Then wash the column to be used with 10 x column volume of MQ.
* Change lanes A1 and B1 into filtered Wash Buffer (30mM NaPi, 500mM NaCl, 20mM imidazole, pH 8.0) and Buffer B (50 mM NaPi, 500 mM NaCl, 500 mM imidazole, pH 7,4). Repeat the same washes as in step 2. Then change lane A1 into Buffer A.
* Make a suitable program for the purification in the software.
* Inject 4 ml sample into the sample loop with a syringe.
* Start the run. Fractions will be collected in tubes/plates.
* After the run wash the machine with filtered MQ, as at the beginning.
* Wash the machine with 20% ethanol. Store the machine in ethanol.
* Select elution fractions with the desired protein from the graph, and run flow-through and elution fractions on an SDS-PAGE. Pool fractions that contain the desired protein, freeze in liquid nitrogen and store at -20°C or -80°C.

Concentration of Proteins Using Sartorius Vivaspin 20 Ultracentrifugation Tubes

* If the Sartorius Vivaspin 20 ultracentrifugation tubes have been stored in ethanol, start by centrifuging for ~45 minutes at 3000 g, until the ethanol has gone through the filter.
* Wash the filter by adding 20 ml of MilliQ -water and centrifuging 30-45 minutes at 3000 g
* Concentrate samples by centrifuging ~30 minutes at a time at 3000 g. Mix samples by pipetting and continue until you reach the wanted volume. Store samples at -80 °C after freezing in liquid nitrogen.
* Clean tubes by washing 2 times with 20 ml Milliq -water at 3000 g for 30-45 min. Store tubes in 20 % ethanol.

Microtitre Plate Growth Experiment

Protocol based on CHEM-E3160, Biolab III -Laboratory Course manual, written by Heli Viskari, Mari Piirainen and Bart Rooijakkers.

* Make a 2-5 ml LB + antibiotic pre-culture from a single colony of each strain. Grow overnight at 37°C
* Add 80 ul of this culture to 4 ml of fresh LB + antibiotic medium, and incubate at 37°C until the OD600 reaches 0.6
* On a flat bottom 96-well plate, Add 10 ul of cells to 200 ml of medium (~ 5 x 10^6 cells to start with). Make three replicates with 0, 0.1, 0.5 and 1 mM IPTG. Controls that can be used are water, growth medium without cells, and growth medium with cells that don’t produce fluorescent protein.
* Run Cytation 3 overnight using following protocol:

+ Set the Temperature to 37°C.
+ Start Kinetics.
+ Shake Orbital for 6 min.
+ Measure OD at 600.
+ Measure Fluorescence (From the bottom of the plate).
+ Measure every 10 minutes.

[![](https://static.igem.org/mediawiki/2018/0/06/T--Aalto-Helsinki--Aalto.png)](http://www.aalto.fi/en/)
[![](https://static.igem.org/mediawiki/2018/1/11/T--Aalto-Helsinki--Helsinki.png)](https://www.helsinki.fi/en)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Aalto-Helsinki/Results

[![](https://static.igem.org/mediawiki/2018/thumb/4/41/T--Aalto-Helsinki--Silkolor.png/1198px-T--Aalto-Helsinki--Silkolor.png)](https://2018.igem.org/Team%3AAalto-Helsinki)

* Project

  [Project Description](https://2018.igem.org/Team%3AAalto-Helsinki/Description)
  [Product Design](https://2018.igem.org/Team%3AAalto-Helsinki/Applied_Design)
* Wet Lab

  [Background](https://2018.igem.org/Team%3AAalto-Helsinki/Background)
  [Experiments](https://2018.igem.org/Team%3AAalto-Helsinki/Experiments)
  [Results](https://2018.igem.org/Team%3AAalto-Helsinki/Demonstrate)
  [InterLab](https://2018.igem.org/Team%3AAalto-Helsinki/InterLab)
* Dry Lab

  [Model](https://2018.igem.org/Team%3AAalto-Helsinki/Model)
* Parts

  [Basic Part](https://2018.igem.org/Team%3AAalto-Helsinki/Basic_Part)
  [Composite Part](https://2018.igem.org/Team%3AAalto-Helsinki/Composite_Part)
* Human Practices

  [Integrated Human Practices](https://2018.igem.org/Team%3AAalto-Helsinki/Human_Practices)
  [Education & Public Engagement](https://2018.igem.org/Team%3AAalto-Helsinki/Public_Engagement)
  [Collaborations](https://2018.igem.org/Team%3AAalto-Helsinki/Collaborations)
* Team

  [Team Members](https://2018.igem.org/Team%3AAalto-Helsinki/Team)
  [Sponsors](https://2018.igem.org/Team%3AAalto-Helsinki/Sponsors)
  [Attributions](https://2018.igem.org/Team%3AAalto-Helsinki/Attributions)
* Achievements

  [Achievements](https://2018.igem.org/Team%3AAalto-Helsinki/Achievements)
  [Judging Form](https://igem.org/2018_Judging_Form?team=Aalto-Helsinki)

# Results

### Cloning We assembled our constructs and cloned them into pSVA1 plasmid using Golden Gate assembly. After transforming plasmids to E.Coli Beta-10 and plasmid isolation we performed a check-up digestion with restriction enzymes NcoI and XhoI which cut the plasmid backbone close to BsaI insert site.

### Protein expression and purification

We were able to successfully purify our silk containing construct prancerPurple-silk-CBM with the heat protocol

### Cellulose binding

Eget duis at tellus at urna. Lacinia quis vel eros donec. Ut pharetra sit amet aliquam id diam. Erat imperdiet sed euismod nisi porta.

### Cellulose nanofibril binding

Eget duis at tellus at urna. Lacinia quis vel eros donec. Ut pharetra sit amet aliquam id diam. Erat imperdiet sed euismod nisi porta.

### Cotton binding

Eget duis at tellus at urna. Lacinia quis vel eros donec. Ut pharetra sit amet aliquam id diam. Erat imperdiet sed euismod nisi porta.

### Binding to hair keratin

Eget duis at tellus at urna. Lacinia quis vel eros donec. Ut pharetra sit amet aliquam id diam. Erat imperdiet sed euismod nisi porta.

Read more

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)

[![](https://static.igem.org/mediawiki/2018/0/06/T--Aalto-Helsinki--Aalto.png)](http://www.aalto.fi/en/)
[![](https://static.igem.org/mediawiki/2018/1/11/T--Aalto-Helsinki--Helsinki.png)](https://www.helsinki.fi/en)