iGEM 2018 — Team UiOslo_Norway

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

# Team:UiOslo Norway/Notebook

Team

[Team members](https://2018.igem.org/Team%3AUiOslo_Norway/Team)
[Collaboration](https://2018.igem.org/Team%3AUiOslo_Norway/Collaborations)

Project

[Description](https://2018.igem.org/Team%3AUiOslo_Norway/Description)
[Design](https://2018.igem.org/Team%3AUiOslo_Norway/Design)
[Results](https://2018.igem.org/Team%3AUiOslo_Norway/Results)
[Demonstrate](https://2018.igem.org/Team%3AUiOslo_Norway/Demonstrate)

Lab

[Experiments](https://2018.igem.org/Team%3AUiOslo_Norway/Experiments)
[Notebook](https://2018.igem.org/Team%3AUiOslo_Norway/Notebook)
[Interlab](https://2018.igem.org/Team%3AUiOslo_Norway/InterLab)

Parts

[Parts Overview](https://2018.igem.org/Team%3AUiOslo_Norway/Parts)
[Basic Parts](https://2018.igem.org/Team%3AUiOslo_Norway/Basic_Part)

Human Practices

[Human Practices](https://2018.igem.org/Team%3AUiOslo_Norway/Human_Practices)
[Integrated Human Practices](https://2018.igem.org/Team%3AUiOslo_Norway/Integrated_Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUiOslo_Norway/Public_Engagement)

Attributions

[Attributions](https://2018.igem.org/Team%3AUiOslo_Norway/Attributions)
[Sponsors](https://2018.igem.org/Team%3AUiOslo_Norway/Sponsors)

[Judging Form](https://igem.org/2018_Judging_Form?team=UiOslo_Norway)

[Medal Criteria](https://2018.igem.org/Team%3AUiOslo_Norway/Medal)

[Safety](https://2018.igem.org/Team%3AUiOslo_Norway/Safety)

Top

# Notebook

Here we have documented our project. This helped us keep track of what has been done, and to document our progress.

## General labwork

Notebook is sorted by calendar week

Week 26

##### sgRNA generator

* Performed transformation of the sgRNA generator from the iGEM distribution kit plate 7, well 20O
* A colony was picked from the transformed sgRNA and inoculated to prepare for a Miniprep.
* Miniprep of sgRNA generator was done.

##### Expression and submission vectors

* A Phusion PCR was done for the pBAD (expression vector) and PSB1C3 (iGEM submission vector) to amplify for later transformations.
* dpnI digestion was done for both vectors.
* PCR purification was then done for both vectors.

Week 27

##### gRNA

* Annealing of gRNA oligos. 4 gRNAs are annealed in total.
* Golden Gate assembly of gRNA oligos and sgRNA generator.
* Transformation was done for the Golden Gate assembly product and plated on chloramphenicol plates with X-Gal and IPTG (for blue white selection) and on regular chloramphenicol and LB medium plates.
* All plates had colonies.
* Overnight cultures were prepared for a later Miniprep.
* A Miniprep was performed for the gRNA.
* gRNAs that was Minipreped was sent for sequencing.

##### dCas9

* dCAS9 constructs arrived from Twist Bioscience.
* A three-part Gibson assembly was performed for dCAS9 constructs A\_i+B\_i (dCas9\_Nlact) and A\_i+B\_ii (dCas9\_Clact).
* Transformation was done for both Gibson assembly’s and plated on chloramphenicol and LB medium plates.

No colonies were observed

* Gibson assembly was repeated for the same constructs.
* A transformation was done from the Gibson assembly and plated on chloramphenicol and LB plates.

Week 28

##### dCas9

* Only the dCas9\_Nlact construct from week 27 gave colonies.
* A Miniprep was done for the dCas9\_Nlact colonies.
* A new Gibson Assembly was performed for both constructs (dCas9\_Nlact and dCas9\_Clact).
* A transformation was done from the Gibson assembly done this week and from week 27 and plated on chloramphenicol and LB plates.
* dCas9\_Nlact that was Minipreped was sent for sequencing

Sequencing of the dCas9\_Nlact showed that it wasn’t the right sequence.

* A new Gibson assembly was performed for both constructs (dCas9\_Nlact and dCas9\_Clact).
* A transformation was done from the Gibson assembly and plated on chloramphenicol and LB plates.

Transformation gave no colonies

* dCas9 constructs were fused together by using the SOE PCR protocol (A\_i+B\_i and A\_i+B\_ii)
* A two-part Gibson assembly was performed with the fused dCas9\_Nlact and dCas9\_Clact constructs.
* A transformation was done from the Gibson assembly and plated on chloramphenicol and LB plates.

Got colonies for both constructs (dCas9\_Nlact and dCas9\_Clact).

* An overnight culture was done for the dCas9\_Nlact and dCas9\_Clact colonies
* A Miniprep was done for the dCas9\_Nlact colonies and dCas9\_Clact.
* We cut open both plasmids f according to the enzyme cutting site reaction give linear plasmids.

##### DH5α cells

* More competent DH5α were made by using the chemically competent DH5α protocol.

Week 29

##### dCas9

* dCas9 constructs were fused together by using the SOE PCR protocol (A\_i+B\_i and A\_i+B\_ii)
* Did an enzyme cutting site reaction for dCas9\_Nlact and dCas9\_Clact from week 28.

##### gRNA

* Did a SpeI digestion of the gRNAs

Results (concentration) after a PCR cleanup didn’t give good enough yield for further use.

* Did a second SpeI digestion of the gRNAs

Results (concentration) after PCR clean up was good enough for further use.

##### Vectors

* RFP-PSB1C3 vector was transformed, plated and later on made into a overnight culture.
* Miniprep was done for the overnight culture.

Week 30

##### Vectors

* Did an amplification of the RFP-PSB1C3 vector according to the Phusion PCR.
* Did a restriction enzyme reaction of the RFP-PSB1C3 to make circular double digested vector.
* PSB1C3 was purified using the gel extraction kit.
* Did a Phusion PCR reaction for the expression vector pBAD.
* Did a gel purification to extract the pBAD vector from a agarose gel.

##### Glucanase

* Glucanase construct arrived from Twist Bioscience.
* A Phusion PCR was done for glucanase.
* Glucanase was digested by using XbaI and SpeI.
* A A3 ligation reaction was done for the glucanase and circular PSB1C3 vector.
* Product from ligation procedure was transformed and plated on LB and chloramphenicol plates.

Got colonies for the glucanase construct.

* Overnight culture and a Miniprep was done from glucanase colonies.
* Glucanase was sent for sequencing for verification.
* Both a Taq-polymerase and a Phusion polymerase reaction were tried for the glucanase construct to check for the right sequence length, but did not give good results on agarose gel.

##### dCas9

* dCas9 constructs were fused together by using the SOE PCR protocol (A\_i+B\_i and A\_i+B\_ii)
* Did a XbaI and SpeI digestion of dCas9\_Nlact from week28.
* An A3 ligation reaction was done for the dCas9\_Nlact and PSB1C3
* Did a two-part Gibson assembly with the dCas9\_Clact construct parts and circular PSB1C3.
* Products from ligation and Gibson assembly was transformed and plated on LB and chloramphenicol plates.

Got colonies for the ligated dCas9\_Nlact and Gibson assembled dCas9\_Clacts constructs.

* Overnight culture and a Miniprep the next day was made for both dCas9 constructs.
* dCas9 constructs was sent for sequencing for verification.
* Did a gradient SOE PCR for the dCas9\_Nlact and dCas9\_Clact constructs to optimize the PCR program for best results.

Did not get good results for this gradient optimization.

* Both a Taq-polymerase and a Phusion polymerase reaction were tried for the dCas9 constructs to check for the right sequence length, but did not give good results on agarose gel.

##### gRNA

* Made gRNA by using our gRNA DNA-templates and Hiscribe kit.
* Removed DNA templates after Hiscribe was done by using optional step in Hiscribe protocol.
* Did a purification of the synthetized RNA by using RNA clean up kit.

Week 31

##### Vectors

* Did a PCR amplification of the pBAD expression vector.

##### Glucanase

* Made overnight cultures of glucanase and did a colony PCR.

Phusion PCR gave good results when checked on gel. Made an overnight culture, did a miniprep and sent for sequencing. Did not give the result wanted.

* More glucanase from plates grown in week 30 was checked by doing a colony PCR

Phusion PCR gave good results when checked on gel. Made an overnight culture, did a miniprep and sent for sequencing. Did not give the results wanted.

##### dCas9

* dCas9\_Nlact and dCas9\_Clact that gave colonies in week 30 was continuously checked by doing a colony PCR.

Phusion PCR of both constructs, gave no good results for any colonies checked.

* Plates with LB and chloramphenicol was divided into grid plates. Colonies from the original transformation in week 30 was streaked out in grids, so that a larger amount of cells could be used for colony PCR.
* Colony PCR was continued, but with a lot more cells (a scop of cells was lysed, instead of a single colony).

No colonies gave the results wanted after phusion PCR.

Week 32

##### Vectors

* psB1C3 was PCR amplified for use in Gibson assembly.
* DpnI digestion of psB1C3 and PCR cleanup.

##### Glucanase

* A Gibson assembly was done with glucanase and psB1C3. A sixfold reaction of insert to vector (1:6) in Gibson assembly was done.
* Gibson assembly was transformed into DHα cells. Growth observed.
* Plates divided into grids was made and colonies from transformation streaked out in separate grids.
* Colony PCR was performed.

Gave good results several colonies. These where made into overnight cultures, minipreped.

##### dCas9

* A colony from the A3 ligation transformation of dCas9\_Nlact gave good results after a phusion PCR checked on gel.

Colony was made into overnight culture, minipreped and sent for sequencing. Did not give the result wanted.

* A Gibson assembly was done for both dCas9\_Nlact and dCas9\_Clact.
* Gibson assembly was transformed into DHα cells. Growth.
* Plates divided into grids was made and colonies from transformation streaked out in separate grids.
* Colony PCR was performed.

No colonies gave results. Positive control works, which indicates that the assembly did not work

* A Gibson assembly was done for both dCas9\_Nlact and dCas9\_Clact.
* Gibson assembly was transformed into DHα cells. Growth.
* Plates divided into grids was made and colonies from transformation streaked out in separate grids.
* Colony PCR was performed.

No colonies gave results. Positive control works, which indicates that the assembly did not work

Week 33

##### Vectors

* Amplification of RFP-psB1C3 was done.
* dpnI digestion of RFP-psB1C3 was done.

##### Glucanase

* XbaI digestion of glucanse miniprep from week 32.

All gave correct sizes when checked on gel.

* Glucanase was sent for sequencing. Sequencing gave the correct insert.

##### dCas9

* A Gibson assembly was done for both dCas9\_Nlact and dCas9\_Clact.
* Gibson assembly was transformed into DHα cells. Growth.
* A Gibson assembly was done for Nlact\_dCas9
* Gibson assembly was transformed into DHα cells. Growth.
* Plates divided into grids was made and colonies from transformation streaked out in separate grids.

Week 34

##### Glucanase

* A PCR was done for the glucanase and afterwards PCR purified.
* A Gibson assembly with glucanase and pBAD (expression vector) was done.
* Gibson assembly was transformed into DHα cells.
* Plates divided into grids was made and colonies from transformation streaked out in separate grids.
* Colony PCR was done. No good results.
* A Gibson assembly with glucanase and pBAD (expression vector) was done.
* Gibson assembly was transformed into DHα cells.

##### dCas9

* A PCR fusion of A\_i+B\_i and A\_i+B\_ii was done by doing a SOE PCR. Worked only for the dCas9\_Nlact.
* Colony PCR of colonies from week 33 was done. No good results.
* A PCR fusion of A\_i+B\_i and A\_i+B\_ii was done by doing a SOE PCR, but changed annealing time at 68oC from 10sec. to 30sec.

Week 35

##### Glucanase

* A colony PCR was done for colonies from week 34.

Gave good results on gel.

* Glucanase colonies was made into overnight cultures, minipreped and sent for sequencing.
* Restriction cutting with NcoI of pBAD-glucanase construct was performed.

##### dCas9

* A Gibson assembly was done for both A\_i+B\_i and A\_i+B\_ii.
* Gibson assembly was transformed into DHα cells.
* Plates divided into grids was made and colonies from transformation streaked out in separate grids.
* A colony PCR was done. No good results.

Week 36

##### Glucanase

* Glucanase was transformed into E.coli BL21 cells.
* Overnight culture was made
* Retrieved *Schizosaccharomyces pombe* and *S. cerevisiae* which was grown to exponential phase before

Week 37

##### Glucanase

* Performed a medium-scale protein expression (with arabinose) and purification
* Ran glucanase on a SDS-gel
* Did a western blot
* Performed a medium-scale protein expression (with arabinose) and purification
* Did an affinity chromatography for induced glucanase mixture. Different colonies and methods gave inconclusive results

Week 38

##### Glucanase

* Checked glucanase fractions on SDS-gel
* Ran a fast protein liquid chromatography (FPLC)

Week 39

##### Glucanase

* Made overnight culture of glucanase
* Performed a medium-scale protein expression (with arabinose) and purification
* pBAD transformed into BL21 cells to use for negative control in glucanase assay
* Performed a plate assay for the glucanase

##### dCas9

* Did a SOE-PCR with A\_i+B\_i(dCas9-Nlact), A\_i+B\_ii(dCas9-Clact) and A\_iii+B\_iii(Nlact-dCas9)
* Checked amplified constructs on gel. No dCas9 constructs was observed amplified.

Week 40

##### Glucanase

* Performed a 96-well plate assay for glucanase (24h incubation). Results were inconclusive
* Performed a 96-well plate assay for glucanase over a shorter time period (4h incubation)
* Performed a 96-well plate assay for glucanase over a shorter time period (1h incubation)

Week 41

##### Submission!

* Shipped our glucanase construct to the iGEM HQ according to submission guidelines (08.10.18)

## Interlab

Notebook is sorted by calendar week

Week 19

##### Transformation of Interlab test devices

We did a transformation of the Interlab test devices 1-6 plus the positive and negative control according to the transformation protocol. After incubation at 37oC overnight we could not observe any colonies.

Week 20

##### Calibration of the plate reader according to the [iGEM protocol](https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf)

OD600 Reference point LUDOX

|  | LUDOX CL-X | H2O |
| --- | --- | --- |
| Replicate 1 | 0,053 | 0,037 |
| Replicate 2 | 0,055 | 0,037 |
| Replicate 3 | 0,055 | 0,037 |
| Replicate 4 | 0,057 | 0,037 |
| Arith. Mean | 0,055 | 0,037 |
| Corrected Abs600 | 0,018 |  |
| Reference OD600 | 0,063 |  |
| OD600/Abs600 | 3,500 |  |

Yielding the following results

![](https://static.igem.org/mediawiki/2018/c/c9/T--UiOslo_Norway--notebook_int1.png)

##### Competency test for DH5α cells

We performed five tests

| CELL TYPE | DNA CONCENTRATION | iGEM TEST KIT |
| --- | --- | --- |
| DH5α | 50 pg/µl | 2017 distribution kit |
| DH5α | 20 pg/µl | 2017 distribution kit |
| DH5α | 100 pg/µl | 2018 distribution kit |
| DH5α | 10 pg/µl | 2018 distribution kit |
| Top10 | 10 pg/µl | 2018 distribution kit |

After incubation at 37oC overnight we could not observe any colonies. Thus, we performed another competency test following the same protocol. No colonies were observed, again.

##### Transformation of Interlab test devices

We did a transformation of the Interlab test devices 1-6 plus the positive and negative control according to the transformation protocol. After incubation at 37oC overnight we could not observe any colonies.

Since both the competency test and the two transformations failed, we concluded that our potentially chemically competent cells possibly aren’t working.

##### Production of chemically competent DH5α cells

We performed the production of chemically competent DH5α cells according to this protocol.

Since the procedure didn’t work we suspected the cell stock we were using was not optimal. We therefore chose to buy new stock of already chemically competent DH5α cells from Thermofisher.

Week 23

##### Transformation of Interlab test devices

We did a transformation of the Interlab test devices 1-6 plus the positive and negative control with the bought chemically competent DH5α cells according to the transformation protocol. After incubation at 37oC overnight we could not observe any colonies.

We did another transformation according to the same protocol. Again we could not observe any colonies.

Week 24

##### Transformation of Interlab test devices

We did a transformation of the Interlab test devices 1-6 plus the positive and negative control with the bought chemically competent DH5α cells according to the iGEM transformation protocol. After incubation at 37oC overnight we could not observe any growth.

Week 26

##### Transformation of Interlab test devices

We did a transformation of the Interlab test devices 1-6 plus the positive and negative control with the bought chemically competent DH5α cells according to the transformation protocol. After incubation at 37oC overnight we could not observe any colonies.

No growth was observed, but the experiment was retried, and we got colonies for each test device and the positive and negative controls. Presumably human error or other factors may have been the cause of this happening.

We decided to contact the iGEM headquarter to see if anyone else had reported problems with the Interlab study, but we discovered that **we had used the wrong wells**. After acknowledging our errors, we were able to start fresh with the correct wells.

##### Always double-check your wells!

Week 27

##### Transformation of Interlab test devices

We did a transformation of the Interlab test devices 1-6 (with the correct wells) plus the positive and negative control with the bought chemically competent DH5α cells according to the transformation protocol. After incubation at 37oC, colonies was observed in all plates.

Week 28

##### Completed the Interlab study

Colonies on the plates from week 27 was re-streaked onto fresh LB-Chloramphenicol plates and grown for a later Miniprep.

Two colonies from each construct plate was inoculated in a overnight culture for a Miniprep the next day

A Miniprep was done according to protocol of each double test devices and negative and positive controls.

The iGEM protocol “Colony forming units per 0.1 OD600 *E.coli* cultures” was performed, and we got the results which is shown in our Interlab page. Colonies on plates where counted the next day of the experiment.

The iGEM protocol “Cell measurement protocol” was performed, and we got the results which is shown in our Interlab page.

##### Flowcytometry

With help from Johannes Landskron, a Flowcytometry study was performed on the same cultures from the cell measurement experiment according to the iGEM protocol.


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

# Team:UiOslo Norway/Experiments

Team

[Team members](https://2018.igem.org/Team%3AUiOslo_Norway/Team)
[Collaboration](https://2018.igem.org/Team%3AUiOslo_Norway/Collaborations)

Project

[Description](https://2018.igem.org/Team%3AUiOslo_Norway/Description)
[Design](https://2018.igem.org/Team%3AUiOslo_Norway/Design)
[Results](https://2018.igem.org/Team%3AUiOslo_Norway/Results)
[Demonstrate](https://2018.igem.org/Team%3AUiOslo_Norway/Demonstrate)

Lab

[Experiments](https://2018.igem.org/Team%3AUiOslo_Norway/Experiments)
[Notebook](https://2018.igem.org/Team%3AUiOslo_Norway/Notebook)
[Interlab](https://2018.igem.org/Team%3AUiOslo_Norway/InterLab)

Parts

[Parts Overview](https://2018.igem.org/Team%3AUiOslo_Norway/Parts)
[Basic Parts](https://2018.igem.org/Team%3AUiOslo_Norway/Basic_Part)

Human Practices

[Human Practices](https://2018.igem.org/Team%3AUiOslo_Norway/Human_Practices)
[Integrated Human Practices](https://2018.igem.org/Team%3AUiOslo_Norway/Integrated_Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUiOslo_Norway/Public_Engagement)

Attributions

[Attributions](https://2018.igem.org/Team%3AUiOslo_Norway/Attributions)
[Sponsors](https://2018.igem.org/Team%3AUiOslo_Norway/Sponsors)

[Judging Form](https://igem.org/2018_Judging_Form?team=UiOslo_Norway)

[Medal Criteria](https://2018.igem.org/Team%3AUiOslo_Norway/Medal)

[Safety](https://2018.igem.org/Team%3AUiOslo_Norway/Safety)

# Experiments

Here you may find detailed information on our experiments and protocols.

## dCas9 and β-lactamase

The sequences for the dCas9-lactamase were too large to be synthesized in one piece.

We therefore received them as separate g-blocks that had to be fused together before we could start expressing the proteins.

Our goal was to fuse the dCas9 with either the C-terminal or N-terminal part of the split β-lactamase.

#### Fusing dCas9-β-lactamase parts

Perform a three-part Gibson assembly with pSB1C3 vector (submission vector), the two dCas9 g-blocks, one already linked to a split-β-lactamase.

Transform the assembly into competent DH5α cells, and let grow overnight to observe colonies.

After colonies are observed, make a gridplate with several of the colonies, followed by making overnight cultures from the gridplate colonies.

Miniprep the overnight cultures to extract the DNA, and send the DNA for sequencing.

#### Expressing and purifying dCas9-β-lactamase

After obtaining colonies with the right DNA sequence, clone the DNA into the pBAD vector (expression vector) using two-part Gibson assembly.

Transform the assembly into competent DH5α cells, and let grow overnight to observe colonies.

After colonies are observed, make a gridplate with several of the colonies, perform colony-PCR, followed by making overnight cultures from the gridplate colonies.

Miniprep the overnight cultures to extract the DNA, and send the DNA for sequencing.

After confirming which colonies have the right DNA sequence, insert the expression vector into a bacterial strain suitable for expression, like BL21, using the transformation protocol.

Make overnight cultures for subsequent small-scale protein expression.

Separate the cell fractions using an SDS gel.

Check protein expression with a Western blot using relevant detection antibodies or conjugates.

Run affinity chromatography of the different fractions using the His-tag, collect the fractions into Eppendorf tubes.

Run the fractions containing your protein of interest on an SDS gel.

## CRISPR guide RNA

Transform the sgRNA generator from team Peking 2015 ([BBa\_K1689000](http://parts.igem.org/Part%3ABBa_K1689000)) into DH5α cells.

After observing colonies from the transformation, make an overnight culture.

Miniprep the overnight cultures to extract the DNA, and send the DNA for sequencing.

The guide RNA oligos were ordered in form of forward and reverse DNA primers from Thermo Fisher.

Anneal the complementary primers and insert the guide RNA-oligo-DNA into the sgRNA generator using Golden Gate assembly.

Transform the Golden Gate assembly product and plate them on LB plates with chloramphenicol, X-gal and IPTG for blue white color selection.

After observing white colonies from the transformation, make an overnight culture.

Miniprep the overnight cultures to extract the DNA, and send the DNA for sequencing.

After confirming which colonies have the right DNA sequence, reverse transcribe the guide RNA-oligo-DNA into guide RNA using HiScribe T7 High Yield RNA Synthesis Kit.

Purify the guide RNA using RNeasy MinElute Cleanup Kit.

## Lytic enzymes

#### Flowthrough of expressing glucanase

Perform a two-part Gibson assembly with glucanase and pSB1C3 vector (submission vector).

Transform the assembly into competent DH5α cells, and let grow overnight to observe colonies.

After colonies are observed, make a gridplate with several of the colonies.

Perform colony-PCR and make overnight cultures for the positive colonies.

Miniprep to purify the DNA.

Send the DNA for sequencing.

After obtaining colonies with the right DNA sequence, insert the DNA into the pBAD vector (expression vector) using two-part Gibson assembly.

Transform the assembly into competent DH5α cells, and let grow overnight to observe colonies.

Miniprep the overnight cultures to extract the DNA, and send the DNA for sequencing.

After confirming which colonies have the right DNA sequence, insert the expression vector into a bacterial strain suitable for expression, like BL21, using the transformation protocol.

Make overnight cultures for subsequent small-scale protein expression/middle-scale protein expression.

Separate the cell fractions using an SDS gel.

Check protein expression with a Western blot using relevant detection antibodies or conjugates.

Run affinity chromatography of the different fractions using their His-tag, collect the fractions into Eppendorf tubes.

Run the fractions containing your protein of interest on an SDS gel.

Use the GlycoSpot-Enzyme Standard assay to test the efficiency of the protein.

## Protocols

Click to view our protocols in PDF-format.

* [Agar plate format with enzyme preparations](https://static.igem.org/mediawiki/2018/a/ae/T--UiOslo_Norway--ProtAgar.pdf)
* [GlycoSpot-Enzyme screening standard](https://static.igem.org/mediawiki/2018/c/ca/T--UiOslo_Norway--ProtGlyco.pdf)
* [Small-scale protein expression](https://static.igem.org/mediawiki/2018/d/d6/T--UiOslo_Norway--ProtMedS.pdf)
* [Medium-scale protein expression and purification (Affinity chromatography)](https://static.igem.org/mediawiki/2018/3/30/T--UiOslo_Norway--ProtMedC.pdf)
* [Medium-scale protein expression and purification (Sonification and plate assay)](https://static.igem.org/mediawiki/2018/d/d1/T--UiOslo_Norway--ProtMedP1.pdf)
* [SDS-PAGE 12%](https://static.igem.org/mediawiki/2018/7/70/T--UiOslo_Norway--ProtSDS.pdf)
* [Western Blot](https://static.igem.org/mediawiki/2018/7/77/T--UiOslo_Norway--ProtWB.pdf)
* [Colony PCR](https://static.igem.org/mediawiki/2018/3/32/T--UiOslo_Norway--Prot1.pdf)
* [DpnI digestion](https://static.igem.org/mediawiki/2018/d/de/T--UiOslo_Norway--Prot2.pdf)
* [Gibson Assembly](https://static.igem.org/mediawiki/2018/1/16/T--UiOslo_Norway--Prot3.pdf)
* [Golden gate assembly](https://static.igem.org/mediawiki/2018/8/85/T--UiOslo_Norway--Prot4.pdf)
* [HiScribe T7 High Yield RNA Synthesis Kit](https://static.igem.org/mediawiki/2018/d/d9/T--UiOslo_Norway--Prot5.pdf)
* [Making chemically competent DH5α cells](https://static.igem.org/mediawiki/2018/5/5f/T--UiOslo_Norway--Prot6.pdf)
* [Miniprep](https://static.igem.org/mediawiki/2018/2/2e/T--UiOslo_Norway--Prot7.pdf)
* [PCR cleanup from Gel](https://static.igem.org/mediawiki/2018/0/05/T--UiOslo_Norway--Prot8.pdf)
* [PCR cleanup](https://static.igem.org/mediawiki/2018/a/a5/T--UiOslo_Norway--Prot9.pdf)
* [Phusion PCR](https://static.igem.org/mediawiki/2018/7/79/T--UiOslo_Norway--Prot10.pdf)
* [SOE splicing by overlap-overhang extension PCR](https://static.igem.org/mediawiki/2018/a/af/T--UiOslo_Norway--Prot11.pdf)
* [Transformation protocol for DH5α](https://static.igem.org/mediawiki/2018/c/cd/T--UiOslo_Norway--Prot12.pdf)

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

# Team:UiOslo Norway/Results

Team

[Team members](https://2018.igem.org/Team%3AUiOslo_Norway/Team)
[Collaboration](https://2018.igem.org/Team%3AUiOslo_Norway/Collaborations)

Project

[Description](https://2018.igem.org/Team%3AUiOslo_Norway/Description)
[Design](https://2018.igem.org/Team%3AUiOslo_Norway/Design)
[Results](https://2018.igem.org/Team%3AUiOslo_Norway/Results)
[Demonstrate](https://2018.igem.org/Team%3AUiOslo_Norway/Demonstrate)

Lab

[Experiments](https://2018.igem.org/Team%3AUiOslo_Norway/Experiments)
[Notebook](https://2018.igem.org/Team%3AUiOslo_Norway/Notebook)
[Interlab](https://2018.igem.org/Team%3AUiOslo_Norway/InterLab)

Parts

[Parts Overview](https://2018.igem.org/Team%3AUiOslo_Norway/Parts)
[Basic Parts](https://2018.igem.org/Team%3AUiOslo_Norway/Basic_Part)

Human Practices

[Human Practices](https://2018.igem.org/Team%3AUiOslo_Norway/Human_Practices)
[Integrated Human Practices](https://2018.igem.org/Team%3AUiOslo_Norway/Integrated_Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUiOslo_Norway/Public_Engagement)

Attributions

[Attributions](https://2018.igem.org/Team%3AUiOslo_Norway/Attributions)
[Sponsors](https://2018.igem.org/Team%3AUiOslo_Norway/Sponsors)

[Judging Form](https://igem.org/2018_Judging_Form?team=UiOslo_Norway)

[Medal Criteria](https://2018.igem.org/Team%3AUiOslo_Norway/Medal)

[Safety](https://2018.igem.org/Team%3AUiOslo_Norway/Safety)

# Results

When working on our detection system in the lab, we used different cloning strategies for different purposes. For example, Gibson cloning was used to clone the endo-1,3-β-glucanase and dCas9-reporter constructs into a vector backbone. To generate the different guide RNAs we made use of Golden Gate cloning. The entire team put a lot of time and effort into the laboratory work (Notebook), but not all our cloning attempts ended up being successful. However, we were able to achieve positive and good results in the end.

## Generation of guide RNA

After using the guide RNA generator from the Peking 2015 team, we verified the constructs by sequencing. RNA was generated with the HiScribe RNA synthesis kit, followed by using a RNA cleanup kit, we were able to produce different guide RNAs. We verified the quality and concentration of the guide RNA using a quBit instrument (Table 1).

**Table 1: Concentration of guide RNA measured with quBit.**The concentration of four different guide RNAs was measured, concentration ranged between 40 ng/µl to 370 ng/µl.

| Guide RNA | Concentration ng/µl |
| --- | --- |
| 1 | 138 |
| 2 | 40 |
| 3 | 370 |
| 4 | 82 |

## Endo-1,3-β-Glucanase

The enzyme glucanase is an important part of our detection system, as it catalyzes the hydrolysis of the β-glucan bond in the yeast cell wall, making it easier to lyse the cell and access the DNA. We were not only able to successfully clone the endo-1,3-β-gluanase into the submission vector (Figure 1) and expression vector (Figure 2), but we were also able it induce its expression (Figure 3). Finally, we could also show the glucanase activity in vitro (Figure 4, Figure 5).

![](https://static.igem.org/mediawiki/2018/4/4d/T--UiOslo_Norway--RFig1.png)

**Figure 1: Colony PCR of glucanase in submission vector pSB1C3**Positive colonies after Two-part Gibson transformation of glucanase g-block (1-15). Glucanase at the size of 1.388 bp (blue line), colony 11 was further used after checking for correct sequence. Ladder Quick-Load 1 kb (N0468S).

![](https://static.igem.org/mediawiki/2018/f/f6/T--UiOslo_Norway--RFig2.png)

**Figure 2: Colony PCR of glucanase in expression vector pBAD**Positive colonies after Two-part Gibson transformation of glucanase (1-9). Glucanase at the size of 1.388 bp plus overhangs (blue line). Ladder Quick-Load 1 kb (N0468S).

The glucanase-pBAD construct was further used for protein expression. Protein expression was induced by adding arabinose (protocols).

## Induction of protein expression

The induction of protein expression was verified after small-/or medium-scale cell culturing, followed by separation of proteins on a 12 % SDS-PAGE (Figure 3). The results show that the endo-1,3-β -glucanase expression was induced after addition of 0.1 % arabinose followed by a 3 h incubation at 30oC (protocol).

![](https://static.igem.org/mediawiki/2018/3/32/T--UiOslo_Norway--RFig3.png)

**Figure 3: SDS-PAGE 12 %**Uninduced (1) and induced samples (2&3) loaded on SDS-PAGE. Glucanase at the size of 52 kDa (red arrow).

## Screening for Polysaccharide Endo-Hydrolases using Insoluble Dyed Polysaccharide

Insoluble dyed substrate is used to detect enzyme activity ([protocol](https://static.igem.org/mediawiki/2018/c/ca/T--UiOslo_Norway--ProtGlyco.pdf)).

![](https://static.igem.org/mediawiki/2018/5/5a/T--UiOslo_Norway--RFig6.png)

**Figure 4: 96-well plate assay to test enzyme activity (Megazyme)**
Absorbance of the glucanase was measured against diluted samples of a standard for enzyme activity (Zymolyase, Nordic Biosite). The glucanase activity was tested with samples from induced cell cultures (crude extract, supernatant and cell pellet). Absorbance was measured 1 h after loading the samples on the 96-well plate.

From Figure 4, it can be seen that the glucanase had an activity in between a 100x and 1.000x dilution of Zymolyase. Of the different cell samples from the induced cell culture, the glucanase from the supernatant had the highest enzymatic activity. We expect the glucanase to have a higher enzymatic activity after further experiments purifying the protein.

![](https://static.igem.org/mediawiki/2018/5/58/T--UiOslo_Norway--RFig5.png)

**Figure 5: Plate assay to display enzyme activity (Glycospot)** Three replicates of assay. Upper left corner ‘crude cell culture’, middle left ‘supernatant’, lower left ‘positive control’ (commercially available enzyme mix), upper right ‘crude diluted’, middle right ‘pellet’, lower right ‘negative control’ (empty pBAD vector). All samples on the left parts of the plates show enzyme activity in all replicates.

## Conclusion

With our results we showed that our BioBrick, the endo- 1,3-β-glucanase, has enzymatic activity and is able to cleave 1,3-β-glucan bonds. The activity is comparable to a 100x-1000x dilution of Zymolyase. Combined with either mechanical force or a detergent, we expect to be able to selectively lyse yeast cell walls.

## Experiments that did not succeed yet

Affinity chromatography of glucanase using FPLC (Fast Protein Liquid Chromatography)
We performed different FPLC (Picture 1-4) with different outcomes. The enzyme was not only found in the expected fractions but also in the flow through.

![](https://static.igem.org/mediawiki/2018/9/9e/T--UiOslo_Norway--FPLC.png)

**Picture 1-4: Absorption spectra from the FPLC of glucanase.** Dark green ‘conductivity’, black ‘imidazole buffer’, light green ‘absorption at 280 nm’, pink ‘absorption at 260 nm’, blue ‘absorption at 214 nm’. Proteins have an expected absorption at 280 nm.

In Picture 1 the glucanase was in fraction A16-21, however, there is also a lot of glucanase present in the flowthrough. In Picture 2 the glucanase is present in fraction A8-10, but again a lot of glucanase was lost in the previous fraction.

In Picture 3 and 4 all the glucanase was released in the flowthrough. We hypothesize that this is due to the column being broken down by the enzymatic activity of the glucanase.

* Although we were not successful cloning the dCas9-beta-lactamase into the submission or expression vector, we went through several different cloning attempts, troubleshooting and optimization strategies.
* For cloning we attempted Gibson assembly, 3A assembly and SOE-PCR. We tried to optimize the protocols using different ratios of substrate, incubation times and gradient PCR.

## Future plans for the project

* Continue trying the clone the dCas9-beta-lactamase in the expression vector for expression and purification
* Test if the guide RNAs-dCas9 construct can detect *C. albicans* DNA
* Purify the glucanase to obtain a purer product.
* Test the glucanase on *C. albicans*
* Develop different guide RNAs to detect a range of pathogens