Notebook

Subtitle

Overview

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Work Space

Our lab and office. Our new home for the summer.

About our lab

The iGEM lab, inside the Applied Sciences building of the TU Delft, has been the place where our science took place. It has the certification for ML-1 experiments, which was enough for our experiments. Here we learned and improved our skills in cloning and manipulating biological materials. We also tried to apply our ideas from paper to the real world. All in all, we discovered the world of synthetic biology from the inside.

About our office

Our office has been our meeting point and our dry-lab zone during the project. Here, amazing talks about biology, engineering and philosophy took place. And here we debated about science and lab topics. To sum up, the office that you can see in the picture in the left has been our home this summer: the place where we built the project, but also where we built our team spirit.

Safety

Lab and Project Safety

Safety in our Lab

Our lab is classified as Level 1 of biosafety. This is the lowest safety level, meaning that our experiments involve low or no risk. The biological materials used for our experiments are handled in an open bench All the members of the team have received safety training, including:

Introduction to sterile working

RNA handling

Laser safety

Microscopy training

ML-1 safety test completion

Chemical safety training

General safety information, regarding contact persons and locations

Computer infrastructure

The safety of our experiments was supervised by Susanne Hage (Safety Manager of the TNW faculty) and Marinka Almering (Safety officer of the TU Delft). The research has been conducted with respect to the regulations of biosafety for The Netherlands, that can be found here

Safety in our Project

Our harmless bacteria produces curli subunits in order to make an inducible biofilm. The curli is natively produced by several safe strains, like the TOP10 used for transformations. Our strain contains a CsgA deletion and can only produce the curli when transformed with the designed plasmid and induced. This part of the project is based on sufficient scientific publications and previous iGEM information and parts. Also, one assay is designed to test the efficiency of the biofilm created carrying a specific affinity tag. For the assay teeth pieces were used, taking care of all the existing regulations and safety recommendations.

The chassis organism used for our project is a modified strain of the lab organism Escherichia coli K-12. It is called E. coli K-12 MG1655 PRO ΔcsgA ompR234.

In addition to our chassis organism, Escherichia coli Top 10 cells were used for cloning procedures

A pig tooth is used for testing our affinity application to hydroxyapatite. This will be arranged following the lab regulations, and the tooth will be provided following all the existing regulations. We will handle the part in the ML1 lab, and using gloves while working with the part. Furthermore, the teeth are cleaned properly before the use. The use of that samples is regulated by the Ministry of Economic affairs, Agriculture and Innovation of the Government of The Netherlands. The waste of all animal byproduct is designated as Specific Animal Waste (following Euralcode 180102). Later on, it is transported to the ZAVIN in Dordrecht for its destruction. For further information, you could contact the Residual Management Department of TU Delft (ReststoffenBeheer@tudelft.nl)

One of the problems to be faced with this project is the production of csgA as biofilm-maker unit. It has been reported previously as a virulence factor from harmful strains of E.coli, because the amyloid make the attachment to surfaces easier. However, we implemented two safety points for minimizing the risk of plasmid transmission to pathogen strains. First of all, the gene which codifies for csgA is preceded by a Rhamnose promoter. That prevents curli (csgA+csgB polymer) to be produces when no inductor is applied to the system. Furthermore, as rhamnose is a metabolizable inductor the gene expression could be avoided just by stopping the rhamnose supply. On the other hand, we aim our project for product testing or industrial production. So, the good laboratory practices and good manufacturing practices in these two fields should avoid any safety issue.

If our project could be fully developed into a real product, it would aim the following fields:

Factories

Lab applications

Consumer products

Test products to be used in the human body

Test products on food

We would like to encourage every interested iGEM team or researcher in general to contact us for further details about the safety in our lab and project!

Day Notes

Subtitle or summary goes here. Should be short - two or three sentences.

Interlab

June

July

August

September

Back to Top

Curli

June

15/06/2015

We started preparing the materials for the lab. All the devices are set up, and the disposables ready to sterilize. At the end of the day, both plastic ware and liquids were autoclaved (121ºC, 15-30 min). Here the materials prepared are summarized:

LB medium, 6 bottles of 200 ml*

LB-agar mixture, 6 bottles of 400 ml*

CaCl2 100mM, 1 bottle of 200 ml*

Ethanol 70%, 1 bottle of 400 ml*

CaCl2 100mM + 40% Glycerol, 1 bottle of 200 ml*

*All the materials were prepared using standard methods

16/06/2015

The first stock of competent cells was made, so the first cloning experiments were conducted. could. The Top10 strain was chosen due to its availability and reliability in the results. 100 tubes of working solution containing 50 µL of Top10 competent cells (1) were made, and stored at -80ºC afterwards.

(1) Protocol for Top10 competent cells

19/06/2015

The cloning experiments started today. The experiments for InterLab study were performed initially (see Interlab labjournal). First, LB-Agar plates were made so they were solid and available for growing the cells later:

*The antibiotics were taken from a stock solution containing:

1:1000 ampicillin

1:1000 chloramphenicol

1:1000 kanamycin

23/06/2015

The following cell cultures were made in 9 mL LB ∆csgA strain, K1316015 and I13504 in and grown overnight at 30°C. I13522 (TETP-GFP) has been taken from the iGEM distribution kit (2015, plate 3, location I6).

Primers were dissolved in sterile Milli-Q water according to the amount specified in the shipping information (3) and shaken at 400 rpm for 20 minutes at room temperature to procedure stock solution for iGEM15 1 and iGEM15 2.

(3): Shipping information for primers.

24/06/2015

Plasmids were isolated for K1316015 using the Promega miniprep unit and the related protocol you can find online (4). Plasmids were stored at minus 20°C (30 µl);

AM35α has been made competent (1) and grown overnight. In order to see whether the density was good enough, the OD was measured.

To check the PCR reaction, an agarose gel (6) was made. The PCR product should be at the level of 2.7 KB.

The construct K1316015∆CsgB was isolated from the gel by using the Promega DNA purification protocol (7).

Finally, the ligation was performed with K1316015∆Csg (0.3 µl, based on a concentration of 153.94 ng/µl).

(4): Protocol related to Promega miniprep unit for plasmid isolation.

(5): Protocol for doing a PCR reaction.

(6): Protocol for running an agarose gel.

(7): Protocol for Promega DNA purification.

25/06/2015

Transformation (2) of ligation product K1316015∆CsgB (top10 cells, 46 ng). After transformation, the cells were plated on LB + CAM.

Plated the transformation products K1316015∆CsgB on LB - Cam plates;

Preparation of competent cells for ∆csgA AM35α. 1 mL of the overnight culture was inoculated in 100 mL of LB medium.

Isolation of genomic csgC & csgEFG with primers Primers selected: iGEM15 15;16;17;18. The primers were dissolved in Milli-Q water (sterile) according to the amount specified in the delivery note (8) and shaken for 20 minutes at 400 rpm at room temperature. Produced primer stock solutions from which the working solutions were prepared (100 µM). By a 1:10 dilution to give a working solution of 10 µM or 10 pmol/µl. Working solutions were produced for a volume of 100 µL.

The competent cells (AM35α) had an OD600 of 0.561 in a volume of 100 mL. Cells were harvested in 2 50mL tubes at 4°C for 5 minutes at 4000 rpm. 10 ml of ice cold 0.1 M CaCl2 was added to each tube to resuspend the cells and incubate them on ice for 20 minutes. They were centrifuged and resuspended in 5 mL ice cold CaCl2 each. Incubation for 1h on ice. The cells were pelleted and resuspended in 5 mL 50mM CaCl2 + 40% glycerol each. They were divided in portions of 50 µL and frozen in liquid N2.

(8): Delivery note of primers.

29/06/2015

Isolation of E.coli U12 DH5α genomic DNA. Cells were grown overnight in 6mL LB and stored in the fridge over the weekend. The genomic DNA was isolated using Promega Wizard genomic DNA purification kit following the producer’s protocol (9). The DNA pellet was rehydrated at 4°C overnight.

Cell cultures were created for the 100 µL plates from 25/06/2015 in 8mL LB+ 80µL 100x AMP (actually, 100 mg/mL). No growth was observed indicating problems with the transformation.

(9): Promega Wizard genomic DNA purification kit.

30/06/2015

Measurements of dsDNA indicates failure of isolation of genomic DNA. The concentration is within the machine’s fluctuation 1-2 ng/µl with fluctuations of 1 ng/µl in measurement.

July

01/07/2015

Transformation of GFP/∆csgB product – plated on LB-CAM; So, plates that should be prepared: 4 plates LB; 2 plates LB-CAM; 6 plates LB-AMP;

Restriction reaction was performed as followed, - ∆csgB was restricted with EcoRI/XbaI (16 µl) The reaction mixtures for restriction were as below and it was incubated at 37°C.

Selected colonies from

∆csgB LB-CAM

I13522 LB-CAM

The selected colonies were incubated in 8 mL of LB containing CAM or AMP and they were grown at 37°C at 210 rpm.

An agarose gel was made, the running was performed at V is 100 mL and for 60 minutes.

02/07/2015

Plasmid isolation (Promega, protocol (4)) of ∆csgB, and I13522. Different in this protocol were the facts that we did not use elution buffer but Milli-Q at 50°C (even if we work with small plasmids). The plasmid concentration was determined with the nanodrop machine. A restriction of these four plasmids was performed at 37°C for 2 hours. To check the restriction products, an agarose gel was performed.

Transformation was performed with Top10 cells prepared by Ilja. The normal protocol of transformation was followed (2) for transforming and pUC19 (as a control). For the transformation 1 µl of DNA was added.

03/07/2015

A gel purification(Promega (7)) and a ligation of ∆csgB and I13522 (to obtain the csgA_GFP construct) were performed. Then, transformation of Ligation product csgA-GFP; Control TOP10 strain; was performed. Cells were plated in duplo and also one control.

06/07/2015

Results of the transformation (csgA-GFP)

1. Empty plasmid control (also growth, 100 colonies);

2. TOP10 + CsgA+GFP (growth, 43 colonies, big).

After the checking the plates, a small culture of 5 mL was made using a single colony from the obtained plates, the following cultures were made: TOP10 + csgA-GFP.

The antibiotics were added in a ratio of 1:1000 (thus in 5 mL, we have added 5µl). After cloning, we put the 50 mL tubes at 37°C at 200 rpm.

Besides that we made LB and LB-agar.

- 2 x 200 mL LB (4 g LB powder and 200 mL MQ-water);

- 1 x 400 mL LB (8 g LB powder and 400 mL MQ-water);

- 2 x 400 mL LB agar, 14.24 LB agar powder and 400 mL MQ-water);

- 1x 900 mL LB agar, 32.05 LB agar powder and 900 mL MQ-water);

Then the materials were autoclaved in the kitchen.

The backbone for our own construct is pSB4K5. For the transformation, we did use the protocol (2). Then we have grown the cells on the plates, I13504 and pUC19 on AMP, J23101, J23106 and J23117 at CAM and pSB4K5 at KAN.

07/07/2015

For IDT transformation we already decided which plates we should use

- 16 plates LB-KAN;

- 16 plates BL-CAM;

08/07/2015

We prepared our own antibiotic stock solutions (although we had to redo it anyway)

Amp: 100mg/ml = 0.1 g/ml (dissolved in water)

Kan: 50 mg/ml = 0.05 mg/ml (dissolved in water)

Cam: 35 mg/ml = 0.035 mg/ml. (dissolved in 100% ethanol).

After preparing the antibiotic stocks in only 1 mL, the supervisors (Essengül) told us to make larger stocks and also filter them in order to sterilize them (since we cannot autoclave them).

13/07/2015

Overnight three tubes with 5mL cultures were grown at 37°C shaken at 200 rpm. These were:

K1316015ΔCsgB

I13522

pSB4K5

14/07/2015

Plasmid isolation of pSB4K5 was done for IDT genes backbones. This was done by using 4,5mL overnight culture for isolation of plasmids, to increase the yield. The yield was determined by using the nanodrop, and the concentration was 62,7 ng/ µL.

Glycerol stock was made from the 0,5mL culture left according to protocol from

I13522, → B1 (F1, block 2, column 1, row 6, box 1)

K1316015ΔCsgB→ B2

15/07/2015

For the gBlocks® we performed the restriction. The standard mixture used was followed.The cutsafe and H2O were mixed in the ratio of 14µl cutsafe and 98µl H2O. The DNA was subscribed as followed (gBlocks®):

Mfp5_CsgA;

CsgA_Mfp3;

CsgA_His;

CsgA;

CsgA_HA;

CsgA_Spytag;

CsgC_low;

CsgC_medium;

CsgC_high;

CsgBC_low;

CsgBC_medium;

CsgBC_high;

CsgEFG;

The restriction was left overnight, the temperature schedule was as follows:

The first 3 hours at 37°C;

20 minutes at 65°C;

The rest of the night at 4°C.

16/07/2015

The restricted IDT genes of 15/07/2015 were ligated according a ratio 3:1 for insert:vector. The first 3h the samples were placed at 16°C and the other 20 minutes it was placed at 4°C. After the ligation, 2µl of each sample was transformed into 50µl TOP10 cells. After 1h at 37°C, 35µl of the sample was plated. For the concentrated sample, we centrifuged the samples for 3 minutes at 4000 rpm. Discarded the supernatant partly and resuspended pellet. Plate also 35µl of concentrated cells.

20/07/2015

Column purification was done on CsgAΔCsgB to remove the small restricted pieces. This was done following protocol of the Wizard column purification.

21/07/2015

CsgEFG was placed in a pSB4K5 plasmid which contains an Kanamycin (KAN) resistance gene. This plasmid was attempted to be grown in Chloramphenicol (CAM), but no growth was obtained. The transformation will be repeated in LB-KAN, with the same colonies used first.

Plasmid isolation was done for cultures #1.1-12.3 (36 total). Analytical restriction was performed to confirm insert size for all samples with EcoRI-HF & PstI-HF (See lab journal, page 38 for pipetted volumes). Only 24 out of 36 had the insert, which will be continued with. The correct plasmids are marked green in Table 1

22/07/2015

Construct 13 has been grown again in LB-KAN. Plasmid isolation was done. These plasmids had to be analytically restricted to make sure that the plasmid contained an insert. To speed up the progress, the analytical restriction (EcoRI-HF & PstI-HF) was done at the same time as the “real” restriction (EcoRI-HF & SpeI-HF).

Restriction of constructs 3-6 were done with EcoRI-HF & XbaI-HF. This should cut out a DNA fragment of around ~10 basepairs, which will be removed using column purification. Restriction of constructs 7-13 were done with EcoRI-HF & SpeI-HF. The restriction products were added to electrophoresis, proceeded for 30 minutes at 120V.

Transformation of I13521 [RFP] into top10 cells. Of the I13521 plasmid, 2µl was added to 50µl of cells, incubated for 30 minutes on ice, heat stocked, punt on ice for 2 minutes and incubated at 37°C in 1 ml of pre-warmed LB for 1 hour. Plated on CAM plates in both low and high concentration. Empty cells were used as control to check the quality of the plates.

Another step we performed was the ligation of CsgA∆CsgB with I13522 (GFP). The concentrations of both plasmids after isolation was equal to CsgA∆CsgB: 3.2 ng/µl and I13522: 5.9 ng/µl. Construct:backbone ratio was equal to 1:3.7.

The reagents were mixed and incubated at room temperature for one hour. After that, the mixture was inactivated by heat shock for ten minutes at 65°C. Half of the ligation mixture (25µl) was used for the transformation. The concentration of the ligation mixture was determined to be 600 ng/µl but this result is rather unlikely and most likely due to the ligation buffer and ligase components.

The restriction described above gave the following results. The cloning of CsgEFG did not work for the 3 colonies chosen; 13.1 and 13.3 were self-ligated plasmids and 13.2 was an empty plasmid. 8.1, 8.2, 8.3, 9.2 and 9.3 were not digested by the restriction enzymes; the only band observed on the gel was the one corresponding to the plasmids.

The restrictions of 10.1, 10.2, 10.3, 11.1, 11.2 and 12.1 were successful. Unfortunately, it was impossible to see the bands under the UV. Therefore, the experiment has to be repeated.

23/07/2015

Material preparation for LB agar. Four new bottles of 400 mL were prepared. 14.24 grams of LB agar mix + 400 mL Milli-Q water were mixed while heating to boiling point. After preparation, the bottles were autoclaved.

Another material preparation was performed, 3 bottles of 200 mL LB and 1 bottle of 400 mL LB were prepared. For the 200 mL bottles, 2 grams of LB powder was weighed and added to 200 mL Milli-Q water and for the other one 4 grams of LB powder was weighted and added to 400 mL Milli-Q water.

Chloramphenicol preparation: 35 mg chloramphenicol was dissolved in 1 mL EtOH 100% to give 1000x concentrated stock.

Besides material preparation, we performed plasmid isolation and after that restriction.

The restricted fragments were put on a 1% Agarose gel. The fragment size of 7 to 9 is equal to 441 bp. However, due to the gel used, these fragments run off the gel. The empty cut plasmid was observed at 2070 bp, but previously analysis showed the insert was there. Both 10 and 12 were observed at the correct fragment size.

Clones for 13 still showed only plasmid self-ligation at 6000 bp. Therefore, the ligation/transformation will be repeated for this one.

Transformation results for the I13521 & CsgA_GFP (I13522)

24/07/2015

Dissolving gel slices and extracting DNA for 10, 11 and 12. After extraction, the following weights for the three types of constructs were obtained.

10: 370 ng;

11: 550 ng;

12: 740 ng.

Membrane binding solution was added in 1:1 and the slice was dissolved at 55°C.

Thus, the samples were loaded on a 2% agarose gel to achieve higher concentrations. For the transformations CsgA_GFP and RFP (I135521) a colony PCR was done, to see if the plasmids contained an insert. We did this by using a sterile tip to pick off a piece of a colony into the PCR reaction mixture. The PCR reaction mixture contained:

We repeated the purification with gel of the restricted plasmid DNA. The gel slices weighted each: 7,2: 900 mg; 8: 600 mg; 9: 420 mg; 10: 500mg; 11: 500 mg; 12: 500 mg;

We dissolved the gel slices in 1ug:1uL membrane binding solution and followed the purification from gel protocol. The final concentrations were: 7.2: 1 ng/µL; 8: 2,5 ng/µL ; 9: 3,2 ng/µL; 10: 2,05 ng/µL; 11: 3,6 ng/µL; 12: 2,5 ng/µL;

The purified DNA fragments were stored in the -20°C.

27/07/2015

When looking on the gel 13 (CsgEFG) did not work. Therefore, the ligation and restriction should be repeated. This is done by restricting the plasmid pSB4K5 (14-07-2015) and CsgEFG (23-07-2015) with EcoRI-HF and PstI-HF, and ligating the products afterwards.

The pSB4K5 was then run on a gel at 110 V for 30 minutes, together with a DNA ladder (Benchtop 1kb DNA ladder) and uncut pSB4K5. In order to make the bands visible on the gel, Blue/orange loading dye was added to the uncut and cut pSB4K5 samples. We looked at the gel using a UV lamp, and cut out the band of the cut pSB4K5 at approximately 1700 kb. Gel purification was then done with a yield of 2.08 ng/µl.

The restricted CsgEFG was column purified using the Wizard SV, gel and PCR clean-up system following protocol with a yield of 1.32 ng/µl.

28/07/2015

Plasmid isolation for CsgA_GFP and I13521 (RFP) in triplicate with the goal to restrict RFP for ligation and confirm the insert for CsgA_GFP. 5 mL of the bacterial cultures were used.

The restrictions of I13521 and CsgA_GFP have been performed EcoRI-HF and SpeI-HF.The restriction has been incubated for 1 hour at 37°C in PCR with final heat shock.

The CsgA has been put on the gel on the left side, RFP on the right. A size of 2400 bp indicates failure of ligation of CsgA with GFP. RFP was confirmed and cut out of the gel.

The DNA constructs made on 22/07/2015 (3.1-6.3) were purified out of the gel. The next step is ligation in a ratio of 3:1 insert:vector. During the ligation, different samples are ligated. The CsgEFG_pSB4K5 and the constructs described in the table above.

Ligation has been incubated for 3 hours at 16°C and the final heat shock, the samples were hold for 6 minutes at 62°C. The improvement in the protocol is the fact that molar ratios are used instead of mass ratios!

Another step performed was the transformation into CaCl2 competent cells. Before the protocol could be followed, the cells were thawed for 20 minutes on ice. After the thawing, 8 µl of the ligation mixture was added to cells and incubated for 30 minutes. Running out of ‘sticks’, some of the results of the transformations were plated the next day.

30/07/2015

The restricted RFP was isolated from the gel, which resulted in concentrations of 17, 14 and 12 ng/µl in volumes of 50µl each. After that, ligation of I13521 (RFP) with CsgA & CsgA_His/HA/SpyTag. The ligation scheme is calculated in such a way that a vector DNA concentration of 50 ng is achieved.

Another part of today was checking the plates made previously (24/07). No colonies were shown there. Consequently, the plates one tested with top 10 cells without plasmid in order to check the KAN antibiotic. So for the plating we used top 10 cells on LB + KAN plates.

31/07/2015

Today we started with plasmid isolation of the overnight cultures 1-20 inoculated at 30/07/2015. For the plasmid isolations, 4.5 mL (3 x 1.5 mL) was used from each overnight cultures, except for 7.1 & 7.3 (3 mL → 2x 1,5 mL) due to pipetting mistake. In the table below, the concentrations we found after the plasmid isolations are shown.

From the obtained plasmids, number 1 to 12 and the Interlab construct J23117_GFP were sent for sequencing. All in a total volume of 10 µl, a total amount of plasmid of 500 ng and a total amount of primer of 25 pmol. The primer working concentration would be 10 pmol/µl.

After plasmid isolation, we performed a restriction reaction. For this, the constructs Mfp5_csgA, CsgA_Mfp3, CsgA_his, CsgA_HA, CsgA and CsgA_Spy (1 to 6) were cutted with the restriction enzymes EcoRI-HF and XbaI-HF. pSB4K5 (14) and pSB1C3 were cut with EcoRI-HF and PstI-HF. Finally, the constructs CsgC_low #1, CsgC_low #2, CsgC_low #3 (7.1, 7.2, 7.3), I13522 #1, I13522 #2, I13522 #3, I13521 #1, I13521 #2, I13521 #3 were cut with EcoRI-HF and SpeI-HF.

The restricted samples were put on a gel to check whether the restriction worked.

- EcoRI/XbaI = small fragment → Column purification for #1-6 (Mfp5_csgA, CsgA_Mfp3, CsgA_his, CsgA_HA, CsgA and CsgA_Spy);

- EcoRI/SpeI = bad of ~700/400bp → Gel purification #9-17 (CsgC_low #1, CsgC_low #2, CsgC_low #3 (7.1, 7.2, 7.3), I13522 #1, I13522 #2, I13522 #3, I13521 #1, I13521 #2, I13521 #3);

- EcoRI/PstI = band of ~3000bp → Gel purification #7-8 (pSB4K5 (14) and pSB1C3).

Column purification was performed according to the protocol. Gel samples #7-17 were loaded with 5µl Blue loading buffer, with 25 µl sample loaded per slot.

The transformation of (30/07) CsgA_His/Spy/””/HA and RFP were grown overnight to confirm the insert, and make a cryostock.

Plasmid isolation was performed for the RFP + CsgA + tags. The samples were lost in the centrifuge so this must be repeated.

Ligation Csg_EFG with pSB1C3 & pSB4K5, CsgA&tags with GFP, Mfp5 & Mfp3 with GFP/RFP.

August

September

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Hardware

June

July

August

September

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Team:TU Delft/Notebook

Notebook

Overview

Work Space

About our lab

About our office

Safety

Safety in our Lab

Safety in our Project

Day Notes

Interlab

Curli

15/06/2015

16/06/2015

19/06/2015

23/06/2015

24/06/2015

25/06/2015

29/06/2015

30/06/2015

01/07/2015

02/07/2015

03/07/2015

06/07/2015

07/07/2015

08/07/2015

13/07/2015

14/07/2015

15/07/2015

16/07/2015

20/07/2015

21/07/2015

22/07/2015

23/07/2015

24/07/2015

27/07/2015

28/07/2015

30/07/2015

31/07/2015

Hardware

Protocols

Gel making

Gel running

Materials

Methodology

Methodology

Methodology

Methodology

Methodology

Characterization of the surface of the samples

Biofilm formation on the surface

Hydroxyapatite-tag strength test

Materials

Preparation bio-ink

Printing

Store

Dissolving gel

Gel making

Resources