Team:UiOslo Norway/Notebook

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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

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

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 37^oC 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 37^oC 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.