Difference between revisions of "Team:Linkoping Sweden/Experiments"

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<div class="column full_size">
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=Experiments=
  
 +
----
  
−
<p>Describe the experiments, research and protocols you used in your iGEM project.</p>
+
==Overview on Laboration==
  
−
</div>
+
===Week 1===
  
−
<div class="column half_size">
+
'''14 June'''
−
<h5>What should this page contain?</h5>
+
−
<ul>
+
−
<li> Protocols </li>
+
−
<li> Experiments </li>
+
−
<li>Documentation of the development of your project </li>
+
−
</ul>
+
  
−
</div>
+
- First day at the lab! Making Hutner’s trace elements.
  
−
<div class="column half_size">
 
−
<h5>Inspiration</h5>
 
−
<ul>
 
−
<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
 
−
<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
 
−
<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
 
−
</ul>
 
−
</div>
 
  
 +
===Week 2===
 +
'''21 June'''
  
−
<div class="clear"></div>
+
- Making SOC medium, LB medium, LB agar and chloramphenicol plates.
  
  
−
<div class="column half_size">
+
===Week 3===
 +
'''27 June'''
  
 +
- Transformation of [http://parts.igem.org/Part:BBa_E1010 BBa_E1010] to test transformation protocol. ''Observation: The transformation was successful.''
  
 +
'''28 June'''
  
−
</div>
+
- Control of competent cells.
  
−
</html>
+
'''29 June'''
 +
 
 +
- Transformation of BBa_E1010 to super competent XL-1. ''Observation: The transformation was successful.''
 +
 
 +
'''30 June'''
 +
 
 +
- Making new ''E.Coli'' Calcium chloride competent cells.
 +
 
 +
'''1 July'''
 +
 
 +
- Making solutions for TAP and TRIS medium.
 +
 
 +
- Cultivation of successful transformants.
 +
 
 +
 
 +
===Week 4===
 +
'''4 July'''
 +
 
 +
- Making LB medium and LB agar.
 +
 
 +
- Plasmid preparation of succeded transformants week 3.
 +
 
 +
- Test cultivation of algae.
 +
 
 +
'''5 July'''
 +
 
 +
- Making agar plates.
 +
 
 +
- Starting standard assembly of [https://2016.igem.org/Team:Linkoping_Sweden/Design construct]; digestion and ligation of pLIP ([http://parts.igem.org/Part:BBa_K2095000 BBa_K2095000]), U6 promoter, RBCS2 terminator ([http://parts.igem.org/Part:BBa_K2095002 K2095002]) and LIP-RFP ([http://parts.igem.org/Part:BBa_K2095003 BBa_K2095003]).
 +
 
 +
- Competent cell test.
 +
 
 +
- First algae cultivation.
 +
 
 +
'''6 July'''
 +
 
 +
- Transformation on U6, pLIP, LIP-RFP and RBCS2 terminator. ''Observation: Colonies for U6, pLIP and LIP-RFP were detected.''
 +
 
 +
'''7 July'''
 +
 
 +
- Cultivation of U6, pLIP, LIP-RFP colonies in LB medium with chloramphenicol. ''Observation: Transformation of insert did not succeed''
 +
 
 +
 
 +
 
 +
 
 +
===Week 5===
 +
'''11 July'''
 +
 
 +
- PCR on Cas9.
 +
 
 +
'''12 July'''
 +
 
 +
- Gel electrophoresis on Cas9 to see if the PCR was successful. ''Observation: No bands were obtained for Cas9.''
 +
 
 +
'''14 July'''
 +
 
 +
- PCR on pSB1C3 for more product.
 +
 
 +
'''15 July'''
 +
 
 +
- Gel electrophoresis on pSB1C3. ''Observation: No bands were obtained on the gel.''
 +
 
 +
 
 +
 
 +
===Week 6===
 +
'''18 July'''
 +
 
 +
- PCR and gel electrophoresis on pSB1C3. ''Observation: No bands were obtained on the gel.''
 +
 
 +
'''20 July'''
 +
 
 +
- PCR and gel electrophoresis on pSB1C3. ''Observation: Correct bands were obtained - PCR on pSB1C3 succeeded.''
 +
 
 +
'''22 July'''
 +
 
 +
- Digestion, ligation and transformation on pLIP, U6, RBCS2 terminator, Cas9, LIP-RFP, sgRNA and pSB1C3.
 +
 
 +
 
 +
===Week 7===
 +
'''25 July'''
 +
 
 +
- PCR and recultivation of transformed pLIP and RBCS2 terminator colonies.
 +
 
 +
- PCR purification of pSB1C3.
 +
 
 +
'''26 July'''
 +
 
 +
- Gel electrophoresis on pSB1C3, pLIP, RBCS2. ''Observation: No bands were obtained on the gel.''
 +
 
 +
- Digestion and ligation on LIP-RFP and pSB1C3.
 +
 
 +
'''27 July'''
 +
 
 +
- '''''New project approach''''' - Left standard assembly for Gibson assembly
 +
 
 +
- Transformation of LIP-RFP and pSB1C3.
 +
 
 +
 
 +
===Week 8===
 +
 
 +
'''1 August'''
 +
 
 +
- Cultivation of hygromycin-containing bacteria in order to isolate hygromycin which would be utilized to test electroporation in ''C.Reinhardtii''
 +
 
 +
- Gel electrophoresis on pLIP and RBCS2 terminator. ''Observation: Bands were obtained on the gel at 700 bp, indicates insert.''
 +
 
 +
'''3 August'''
 +
 
 +
- Plasmid preparation of pLIP, RBCS2 terminator and hygromycin. ''Observation: Turned out to be incorrect later on.''
 +
 
 +
- Transformation of LIP-RFP, U6, sgRNA and Cas9. ''Observation: 4 colonies for sgRNA and 4 colonies for LIP-RFP were obtained, but unfortunately no colonies for Cas9 and U6.''
 +
 
 +
'''4 August'''
 +
 
 +
- Making TAP medium for cultivation of algae in the dark.
 +
 
 +
 
 +
===Week 9===
 +
 
 +
'''8 August'''
 +
 
 +
- PCR and recultivation on transformed LIP-RFP and sgRNA colonies.
 +
 
 +
- First algae cultivation in darkness.
 +
 
 +
'''9 August'''
 +
 
 +
- Transformation of RBCS2 terminator and Cas9.
 +
 
 +
- Gel electrophoresis on LIP-RFP and sgRNA. ''Observation: No bands were obtained.''
 +
 
 +
'''10 August'''
 +
 
 +
- PCR on LIP-RFP and sgRNA.
 +
 
 +
- Gel electrophoresis on LIP-RFP. ''Observation: Bands slightly above 1000 bp were obtained. LIP-RFP with primers should be at 1300 bp. It is looking good.'' 
 +
 
 +
'''11 August'''
 +
 
 +
- PCR on U6 and Cas9.
 +
 
 +
- Gel electrophoresis on sgRNA. ''Observation: No correct bands were obtained.''
 +
 
 +
'''12 August'''
 +
 
 +
- Gel electrophoresis on Cas9 and U6. ''Observation: No correct bands were obtained.''
 +
 
 +
 
 +
===Week 10===
 +
'''15 August'''
 +
 
 +
- Preparation of TAP agar. ''Observation: Because of difficulties with the gas no plates could be performed today.''
 +
 
 +
- Gel electrophoresis on RBCS2 terminator, pLIP, hygromycin and pSB1C3. ''Observation: No correct bands were obtained except for pSB1C3.''
 +
 
 +
- New cultivation of hygromycin-containing bacteria.
 +
 
 +
'''16 August'''
 +
 
 +
- Cultivation of sgRNA, LIP-RFP and U6 transformants.
 +
 
 +
- PCR on Cas9 and hygromycin.
 +
 
 +
- Gel electrophoresis on Cas9 and hygromycin. ''Observation: The gel showed a weak band for Cas9 around 4000 bp.''
 +
 
 +
'''17 August'''
 +
 
 +
- Plasmid preparation of LIP-RFP, U6 and sgRNA. ''Observation: Turned out to be incorrect later on.''
 +
 
 +
'''18 August'''
 +
 
 +
- Cultivation of algae for transformation. ''Observation: It took 5 days for the wild type algae to reach OD = 1,757. The mutant algae evaporated.''
 +
 
 +
- Making TAP agar plates.
 +
 
 +
- Making TAP Hyg. plates.
 +
 
 +
 
 +
===Week 11===
 +
 
 +
'''22 August'''
 +
 
 +
- Cultivation of LIP-RFP and hygromycin.
 +
 
 +
'''23 August'''
 +
 
 +
- Plasmid preparation of LIP-RFP and hygromycin-containing bacteria.
 +
 
 +
- PCR on Cas9 and pSB1C3.
 +
 
 +
- New cultivation of algae in the dark.
 +
 
 +
'''24 August'''
 +
 
 +
- PCR on LIP-RFP, hygromycin and pSB1C3.
 +
 
 +
- Gel electrophoresis on Cas9, hygromycin, LIP-RFP and pSB1C3. ''Observation: Bands for Cas9 and hygromycin were obtained.''
 +
 
 +
- PCR purification of Cas9.
 +
 
 +
'''25 August'''
 +
 
 +
- Gel electrophoresis on pSB1C3. ''Observation: Bands were detected at 2000 bp which corresponds to the size of pSB1C3.''
 +
 
 +
- PCR purification of pSB1C3.
 +
 
 +
- '''''First Gibson Assembly!'''''
 +
 
 +
- Transformation of Gibson Assembly. ''Observation: Colonies were obtained!''
 +
 
 +
- PCR on Cas9, hygromycin, pSB1C3.
 +
 
 +
'''26 August'''
 +
 
 +
- PCR and recultivation of Gibson Assembly transformants.
 +
 
 +
- Chloramphenicol plates.
 +
 
 +
- Gel electrophoresis on PCR product from Gibson Assembly, Cas9, hygromycin and pSB1C3. ''Observation: Bands were detected for all the DNAs!''
 +
 
 +
 
 +
===Week 12===
 +
'''29 August'''
 +
 
 +
- Gel electrophoresis on Gibson Assembly colonies. ''Observation: Band at 5500 bp was obtained, 7000 bp would indicate complete insert.''
 +
 
 +
- A new digestion on LIP-RFP.
 +
 
 +
- PCR screening on Gibson Assembly colonies.
 +
 
 +
'''30 August'''
 +
 
 +
- PCR screening on Gibson Assembly colonies.
 +
 
 +
- Cultivation of Gibson Assembly colonies on new plates.
 +
 
 +
- Ligation and transformation on LIP-RFP with pSB1C3.
 +
 
 +
- New Gibson Assembly transformation.
 +
 
 +
'''31 August'''
 +
 
 +
- Gel electrophoresis on Gibson Assembly colonies. ''Observation: The bands indicate that there is no insert.''
 +
 
 +
- Plasmid preparation of Gibson Assembly colony (2016-08-29).
 +
 
 +
'''1 September'''
 +
 
 +
- PCR on plasmid prepared U6, RBCS2 terminator, pLIP, LIP-RFP, sgRNA and hygromycin.
 +
 
 +
- Gel electrophoresis on  U6, RBCS2 terminator, pLIP, LIP-RFP, sgRNA and hygromycin ''Observation: No bands were obtained.''
 +
 
 +
'''2 September'''
 +
 
 +
- Second Gibson Assembly.
 +
 
 +
- Gibson transformation.
 +
 
 +
- Transformation of LIP-RFP.
 +
 
 +
'''3 September'''
 +
 
 +
- PCR  and gel electrophoresis on Gibson colonies. ''Observation: No bands were obtained on the gel.''
 +
 
 +
- Digestion and ligation of pLIP, U6, RBCS2 terminator, sgRNA and pSB1C3.
 +
 
 +
'''4 September'''
 +
 
 +
- PCR and gel electrophoresis on Gibson colonies. ''Observation: Band from one colony (colony 8) indicates correct insert!''
 +
 
 +
 
 +
===Week 13===
 +
'''5 September'''
 +
 
 +
- PCR on old colonies of LIP-RFP.
 +
 
 +
- Making LB medium.
 +
 
 +
- Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies.
 +
 
 +
'''6 September'''
 +
 
 +
- Cultivation of colony 8 (Gibson Assembly) with hygromycin in the LB media.
 +
 
 +
- Screening of colonies from Gibson Assembly. ''Observation: Bands were obtained, but no band was at 7000 bp.''
 +
 
 +
'''7 September'''
 +
 
 +
- PCR and gel electrophoresis on hygromycin.
 +
 
 +
'''8 September'''
 +
 
 +
- Plasmid preparation of Gibson Assembly colony 8.
 +
 
 +
- Screening on Gibson colonies.
 +
 
 +
'''9 September'''
 +
 
 +
'''''The sequence from colony 8 was obtained.''''' ''We did not insert hygromycin but instead YFP was inserted, this due to a 50% chance of amplifying either hygromycin or YFP when using PCR. Cas9 and pLIP were inserted successfully!''
 +
 
 +
'''10 September'''
 +
 
 +
- Cultivation of algae mutants and Gibson colony 8.
 +
 
 +
- Gel electrophoresis on Gibson colonies and plasmid prepared colony 8. ''Observation: The plasmid preparation of Gibson colony 8 showed good bands.''
 +
 
 +
'''11 September'''
 +
 
 +
- PCR on Gibson colonies.
 +
 
 +
- Recultivation of Gibson Assembly colonies on new plates.
 +
 
 +
 
 +
===Week 14===
 +
'''13 September'''
 +
 
 +
- OD measurements on the algae.
 +
 
 +
- Gel electrophoresis on the PCR product from 11/9 - 16. ''Observation: No correct bands were obtained.''
 +
 
 +
'''14 September'''
 +
 
 +
- Dilution of the algae.
 +
 
 +
- Making TAP-40mM sucrose.
 +
 
 +
- Plasmid preparation of Gibson Assembly colony 8.
 +
 
 +
'''15 September'''
 +
 
 +
- Digestion of Gibson colony 8, for the electroporation.
 +
 
 +
'''16 September'''
 +
 
 +
- Electroporation on algae with Gibson colony 8 as the vector ''Observation: The algae have grown well.''
 +
 
 +
'''17 September'''
 +
 
 +
- PCR screening on Gibson colonies.
 +
 
 +
- Continuation on the electroporation from previous day.
 +
 
 +
'''18 September'''
 +
 
 +
- Gel electrophoresis on Gibson colonies from previous day.
 +
 
 +
 
 +
===Week 15===
 +
'''19 September'''
 +
 
 +
- The sequence from Gibson colony 8 was obtained. ''Observation: Looks like we did not insert U6 and sgRNA.''
 +
 
 +
'''20 September'''
 +
 
 +
- Third Gibson Assembly.
 +
 
 +
- Gibson transformation.
 +
 
 +
'''21 September'''
 +
 
 +
- PCR screening on the third Gibson Assembly.
 +
 
 +
- Gel electrophoresis on the PCR product from today. ''Observation: Band were obtained at 300 bp and 2000 bp.''
 +
 
 +
'''22 September'''
 +
 
 +
- Gel electrophoresis on PCR product from previous day. ''Observation: No correct bands were obtained.''
 +
 
 +
- A new Gibson Assembly but only on pSB1C3 with pLIP, LIP-RFP, U6 and RBCS2 terminator respectively.
 +
 
 +
- Gibson transformation.
 +
 
 +
'''23 September'''
 +
 
 +
- Gel electrophoresis on the obtained colonies from the third Gibson. ''Observation: No bands were detected on the gel.''
 +
 
 +
- PCR of Gibson with pLIP, LIP-RFP, U6 and RBCS2 terminator.
 +
 
 +
- Screening of YFP transformed algae. ''Observation: No proof that the transformation worked.''
 +
 
 +
'''24 September'''
 +
 
 +
- Gel electrophoresis on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. ''Observation: Bands were obtained at 500 bp for RBCS2 terminator, 600 bp for pLIP and 1500 bp for LIP-RFP. No result for U6.''
 +
 
 +
- Gel electrophoresis on the colonies obtained from the third Gibson. ''Observation: The bands indicate that there was no insert.''
 +
 
 +
- PCR on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP.
 +
 
 +
- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.
 +
 
 +
'''25 September'''
 +
 
 +
- PCR on the colonies obtained from the third Gibson.
 +
 
 +
- Gel electrophoresis on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. ''Observation: Bands were obtained for all the DNA fragments except from U6''
 +
 
 +
- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.
 +
 
 +
 
 +
===Week 16===
 +
'''26 September'''
 +
 
 +
- PCR on U6 colonies.
 +
 
 +
- Gel electrophoresis on the colonies obtained from the third Gibson. ''Observation: The bands indicate that there is no insert.''
 +
 
 +
- Plasmid preparation of pLIP, LIP-RFP and RBCS2 terminator.
 +
 
 +
'''27 September'''
 +
 
 +
- Plasmid preparation nr 2 of pLIP, LIP-RFP and RBCS2 terminator.
 +
 
 +
- Gel electrophoresis on U6 colonies. ''Observation: No results.''
 +
 
 +
- PCR on the colonies obtained from the third Gibson.
 +
 
 +
'''28 September'''
 +
 
 +
- Gel electrophoresis on the colonies obtained from the third Gibson. ''Observation: No results.''
 +
 
 +
- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.
 +
 
 +
- PCR on the colonies obtained from the third Gibson.
 +
 
 +
'''29 September'''
 +
 
 +
- Gel electrophoresis on the PCR product from the previous day. ''Observation: The bands indicate that there is no insert.''
 +
 
 +
'''1 October'''
 +
 
 +
- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. ''Observation: Correct bands were obtained!.''
 +
 
 +
- Plasmid preparation of pLIP, LIP-RFP and RBCS2 terminator.
 +
 
 +
- New cultivation of pLIP on plates.
 +
 
 +
'''2 October'''
 +
 
 +
- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. ''Observation: Correct bands were obtained!''
 +
 
 +
 
 +
===Week 17===
 +
'''3 October'''
 +
 
 +
- Sequencing of pLIP, LIP-RFP and RBCS2 terminator.
 +
 
 +
'''5 October'''
 +
 
 +
- New Gibson Assembly on pSB1C3 with U6.
 +
 
 +
- Gibson transformation on pSB1C3 with U6.
 +
 
 +
'''6 October'''
 +
 
 +
- PCR on U6.
 +
 
 +
- Cultivation of LIP-RFP.
 +
 
 +
'''7 October'''
 +
 
 +
- Gel electrophoresis on U6. ''Observation: The band indicate that there is no insert.''
 +
 
 +
- Cultivation of algae.
 +
 
 +
'''8 October'''
 +
 
 +
- Cultivation of LIP-RFP.
 +
 
 +
'''9 October'''
 +
 
 +
- Plasmid preparation of LIP-RFP.
 +
 
 +
 
 +
===Week 18===
 +
'''11 October'''
 +
 
 +
- Plasmid preparation of pLIP and RBCS2 terminator.
 +
 
 +
'''12 October'''
 +
 
 +
- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. '''''This was the last day at the lab!'''''
 +
 
 +
===Week 19===
 +
'''19 October'''
 +
 
 +
- Parts were finally prepared for submission!
 +
 
 +
'''20 October'''
 +
- Parts submitted
 +
 
 +
 
 +
{{Linkoping_Sweden/Footer}}

Latest revision as of 17:46, 19 October 2016

LiU iGEM
Team
Collaborations Attributions
Project
Design Experiments Protocols Results Safety Notebook Economic Viability
Project Description
CRISPR/Cas9 System Gibson Assembly Chlamydomonas Reinhardtii
Parts
Human Practices
Silver Gold

Experiments

Overview on Laboration

Week 1

14 June

- First day at the lab! Making Hutner’s trace elements.


Week 2

21 June

- Making SOC medium, LB medium, LB agar and chloramphenicol plates.


Week 3

27 June

- Transformation of [http://parts.igem.org/Part:BBa_E1010 BBa_E1010] to test transformation protocol. Observation: The transformation was successful.

28 June

- Control of competent cells.

29 June

- Transformation of BBa_E1010 to super competent XL-1. Observation: The transformation was successful.

30 June

- Making new E.Coli Calcium chloride competent cells.

1 July

- Making solutions for TAP and TRIS medium.

- Cultivation of successful transformants.


Week 4

4 July

- Making LB medium and LB agar.

- Plasmid preparation of succeded transformants week 3.

- Test cultivation of algae.

5 July

- Making agar plates.

- Starting standard assembly of construct; digestion and ligation of pLIP ([http://parts.igem.org/Part:BBa_K2095000 BBa_K2095000]), U6 promoter, RBCS2 terminator ([http://parts.igem.org/Part:BBa_K2095002 K2095002]) and LIP-RFP ([http://parts.igem.org/Part:BBa_K2095003 BBa_K2095003]).

- Competent cell test.

- First algae cultivation.

6 July

- Transformation on U6, pLIP, LIP-RFP and RBCS2 terminator. Observation: Colonies for U6, pLIP and LIP-RFP were detected.

7 July

- Cultivation of U6, pLIP, LIP-RFP colonies in LB medium with chloramphenicol. Observation: Transformation of insert did not succeed



Week 5

11 July

- PCR on Cas9.

12 July

- Gel electrophoresis on Cas9 to see if the PCR was successful. Observation: No bands were obtained for Cas9.

14 July

- PCR on pSB1C3 for more product.

15 July

- Gel electrophoresis on pSB1C3. Observation: No bands were obtained on the gel.


Week 6

18 July

- PCR and gel electrophoresis on pSB1C3. Observation: No bands were obtained on the gel.

20 July

- PCR and gel electrophoresis on pSB1C3. Observation: Correct bands were obtained - PCR on pSB1C3 succeeded.

22 July

- Digestion, ligation and transformation on pLIP, U6, RBCS2 terminator, Cas9, LIP-RFP, sgRNA and pSB1C3.


Week 7

25 July

- PCR and recultivation of transformed pLIP and RBCS2 terminator colonies.

- PCR purification of pSB1C3.

26 July

- Gel electrophoresis on pSB1C3, pLIP, RBCS2. Observation: No bands were obtained on the gel.

- Digestion and ligation on LIP-RFP and pSB1C3.

27 July

- New project approach - Left standard assembly for Gibson assembly

- Transformation of LIP-RFP and pSB1C3.


Week 8

1 August

- Cultivation of hygromycin-containing bacteria in order to isolate hygromycin which would be utilized to test electroporation in C.Reinhardtii

- Gel electrophoresis on pLIP and RBCS2 terminator. Observation: Bands were obtained on the gel at 700 bp, indicates insert.

3 August

- Plasmid preparation of pLIP, RBCS2 terminator and hygromycin. Observation: Turned out to be incorrect later on.

- Transformation of LIP-RFP, U6, sgRNA and Cas9. Observation: 4 colonies for sgRNA and 4 colonies for LIP-RFP were obtained, but unfortunately no colonies for Cas9 and U6.

4 August

- Making TAP medium for cultivation of algae in the dark.


Week 9

8 August

- PCR and recultivation on transformed LIP-RFP and sgRNA colonies.

- First algae cultivation in darkness.

9 August

- Transformation of RBCS2 terminator and Cas9.

- Gel electrophoresis on LIP-RFP and sgRNA. Observation: No bands were obtained.

10 August

- PCR on LIP-RFP and sgRNA.

- Gel electrophoresis on LIP-RFP. Observation: Bands slightly above 1000 bp were obtained. LIP-RFP with primers should be at 1300 bp. It is looking good.

11 August

- PCR on U6 and Cas9.

- Gel electrophoresis on sgRNA. Observation: No correct bands were obtained.

12 August

- Gel electrophoresis on Cas9 and U6. Observation: No correct bands were obtained.


Week 10

15 August

- Preparation of TAP agar. Observation: Because of difficulties with the gas no plates could be performed today.

- Gel electrophoresis on RBCS2 terminator, pLIP, hygromycin and pSB1C3. Observation: No correct bands were obtained except for pSB1C3.

- New cultivation of hygromycin-containing bacteria.

16 August

- Cultivation of sgRNA, LIP-RFP and U6 transformants.

- PCR on Cas9 and hygromycin.

- Gel electrophoresis on Cas9 and hygromycin. Observation: The gel showed a weak band for Cas9 around 4000 bp.

17 August

- Plasmid preparation of LIP-RFP, U6 and sgRNA. Observation: Turned out to be incorrect later on.

18 August

- Cultivation of algae for transformation. Observation: It took 5 days for the wild type algae to reach OD = 1,757. The mutant algae evaporated.

- Making TAP agar plates.

- Making TAP Hyg. plates.


Week 11

22 August

- Cultivation of LIP-RFP and hygromycin.

23 August

- Plasmid preparation of LIP-RFP and hygromycin-containing bacteria.

- PCR on Cas9 and pSB1C3.

- New cultivation of algae in the dark.

24 August

- PCR on LIP-RFP, hygromycin and pSB1C3.

- Gel electrophoresis on Cas9, hygromycin, LIP-RFP and pSB1C3. Observation: Bands for Cas9 and hygromycin were obtained.

- PCR purification of Cas9.

25 August

- Gel electrophoresis on pSB1C3. Observation: Bands were detected at 2000 bp which corresponds to the size of pSB1C3.

- PCR purification of pSB1C3.

- First Gibson Assembly!

- Transformation of Gibson Assembly. Observation: Colonies were obtained!

- PCR on Cas9, hygromycin, pSB1C3.

26 August

- PCR and recultivation of Gibson Assembly transformants.

- Chloramphenicol plates.

- Gel electrophoresis on PCR product from Gibson Assembly, Cas9, hygromycin and pSB1C3. Observation: Bands were detected for all the DNAs!


Week 12

29 August

- Gel electrophoresis on Gibson Assembly colonies. Observation: Band at 5500 bp was obtained, 7000 bp would indicate complete insert.

- A new digestion on LIP-RFP.

- PCR screening on Gibson Assembly colonies.

30 August

- PCR screening on Gibson Assembly colonies.

- Cultivation of Gibson Assembly colonies on new plates.

- Ligation and transformation on LIP-RFP with pSB1C3.

- New Gibson Assembly transformation.

31 August

- Gel electrophoresis on Gibson Assembly colonies. Observation: The bands indicate that there is no insert.

- Plasmid preparation of Gibson Assembly colony (2016-08-29).

1 September

- PCR on plasmid prepared U6, RBCS2 terminator, pLIP, LIP-RFP, sgRNA and hygromycin.

- Gel electrophoresis on U6, RBCS2 terminator, pLIP, LIP-RFP, sgRNA and hygromycin Observation: No bands were obtained.

2 September

- Second Gibson Assembly.

- Gibson transformation.

- Transformation of LIP-RFP.

3 September

- PCR and gel electrophoresis on Gibson colonies. Observation: No bands were obtained on the gel.

- Digestion and ligation of pLIP, U6, RBCS2 terminator, sgRNA and pSB1C3.

4 September

- PCR and gel electrophoresis on Gibson colonies. Observation: Band from one colony (colony 8) indicates correct insert!


Week 13

5 September

- PCR on old colonies of LIP-RFP.

- Making LB medium.

- Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies.

6 September

- Cultivation of colony 8 (Gibson Assembly) with hygromycin in the LB media.

- Screening of colonies from Gibson Assembly. Observation: Bands were obtained, but no band was at 7000 bp.

7 September

- PCR and gel electrophoresis on hygromycin.

8 September

- Plasmid preparation of Gibson Assembly colony 8.

- Screening on Gibson colonies.

9 September

The sequence from colony 8 was obtained. We did not insert hygromycin but instead YFP was inserted, this due to a 50% chance of amplifying either hygromycin or YFP when using PCR. Cas9 and pLIP were inserted successfully!

10 September

- Cultivation of algae mutants and Gibson colony 8.

- Gel electrophoresis on Gibson colonies and plasmid prepared colony 8. Observation: The plasmid preparation of Gibson colony 8 showed good bands.

11 September

- PCR on Gibson colonies.

- Recultivation of Gibson Assembly colonies on new plates.


Week 14

13 September

- OD measurements on the algae.

- Gel electrophoresis on the PCR product from 11/9 - 16. Observation: No correct bands were obtained.

14 September

- Dilution of the algae.

- Making TAP-40mM sucrose.

- Plasmid preparation of Gibson Assembly colony 8.

15 September

- Digestion of Gibson colony 8, for the electroporation.

16 September

- Electroporation on algae with Gibson colony 8 as the vector Observation: The algae have grown well.

17 September

- PCR screening on Gibson colonies.

- Continuation on the electroporation from previous day.

18 September

- Gel electrophoresis on Gibson colonies from previous day.


Week 15

19 September

- The sequence from Gibson colony 8 was obtained. Observation: Looks like we did not insert U6 and sgRNA.

20 September

- Third Gibson Assembly.

- Gibson transformation.

21 September

- PCR screening on the third Gibson Assembly.

- Gel electrophoresis on the PCR product from today. Observation: Band were obtained at 300 bp and 2000 bp.

22 September

- Gel electrophoresis on PCR product from previous day. Observation: No correct bands were obtained.

- A new Gibson Assembly but only on pSB1C3 with pLIP, LIP-RFP, U6 and RBCS2 terminator respectively.

- Gibson transformation.

23 September

- Gel electrophoresis on the obtained colonies from the third Gibson. Observation: No bands were detected on the gel.

- PCR of Gibson with pLIP, LIP-RFP, U6 and RBCS2 terminator.

- Screening of YFP transformed algae. Observation: No proof that the transformation worked.

24 September

- Gel electrophoresis on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. Observation: Bands were obtained at 500 bp for RBCS2 terminator, 600 bp for pLIP and 1500 bp for LIP-RFP. No result for U6.

- Gel electrophoresis on the colonies obtained from the third Gibson. Observation: The bands indicate that there was no insert.

- PCR on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP.

- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.

25 September

- PCR on the colonies obtained from the third Gibson.

- Gel electrophoresis on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. Observation: Bands were obtained for all the DNA fragments except from U6

- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.


Week 16

26 September

- PCR on U6 colonies.

- Gel electrophoresis on the colonies obtained from the third Gibson. Observation: The bands indicate that there is no insert.

- Plasmid preparation of pLIP, LIP-RFP and RBCS2 terminator.

27 September

- Plasmid preparation nr 2 of pLIP, LIP-RFP and RBCS2 terminator.

- Gel electrophoresis on U6 colonies. Observation: No results.

- PCR on the colonies obtained from the third Gibson.

28 September

- Gel electrophoresis on the colonies obtained from the third Gibson. Observation: No results.

- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.

- PCR on the colonies obtained from the third Gibson.

29 September

- Gel electrophoresis on the PCR product from the previous day. Observation: The bands indicate that there is no insert.

1 October

- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. Observation: Correct bands were obtained!.

- Plasmid preparation of pLIP, LIP-RFP and RBCS2 terminator.

- New cultivation of pLIP on plates.

2 October

- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. Observation: Correct bands were obtained!


Week 17

3 October

- Sequencing of pLIP, LIP-RFP and RBCS2 terminator.

5 October

- New Gibson Assembly on pSB1C3 with U6.

- Gibson transformation on pSB1C3 with U6.

6 October

- PCR on U6.

- Cultivation of LIP-RFP.

7 October

- Gel electrophoresis on U6. Observation: The band indicate that there is no insert.

- Cultivation of algae.

8 October

- Cultivation of LIP-RFP.

9 October

- Plasmid preparation of LIP-RFP.


Week 18

11 October

- Plasmid preparation of pLIP and RBCS2 terminator.

12 October

- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. This was the last day at the lab!

Week 19

19 October

- Parts were finally prepared for submission!

20 October - Parts submitted


LiU iGEM is proudly sponsored by

LiU iGEM
Kårallen, Linköpings Universitet
581 83 Linköping

liuigemgroup@gmail.com