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<li><span class="activiteit">Protein expression of bacteria containing MCS-1 and bacteria containing MCS-1 with Neongreen </span></li>
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<li><span class="activiteit">FACS (labelling bacteria with DBCO-PEG4-tamra) shows a click reaction with bacteria containing MCS-1 &amp; MCS-2. No click reaction occurs with bacteria containing only MCS-1</span></li>
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<li><span class="activiteit">Transform of the newly created plasmid MCS-1 (OmpX-NanoLuc) and MCS-2 (OmpX-neongreen)</span></li>
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<li><span class="activiteit">Protein expression of constructs with mNeongreen and Nanoluc</span></li>
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<li><span class="activiteit">Colony PCR</span></li>
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<li><span class="activiteit">Luminescence and fluorescence assays of expressed proteins</span></li>
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<li><span class="activiteit">Verification of protein expression using a 10% SDS-PAGE gel</span></li>
 
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<span class="activiteit">Gibson Assembly:</span>
 
<span class="activiteit">Gibson Assembly:</span>
 
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<li><span class="activiteit">Succesfull double transformation of 12.2 at 20 ng/uL (rather than 4)</span></li>
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<li><span class="activiteit">Succesfull double transformation of construct 14</span></li>
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<li><span class="activiteit">Gibson Assembly, Colony PCR and transformation of NeonGreen into MCS1</span></li>
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<li><span class="activiteit">Sequencing of the constructs</span></li>
 
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Revision as of 09:09, 6 August 2015





Timeline



Week 26

- Preparing for take-off

  • Inventory of the lab supplies
  • Pouring LB Agar plates
  • Amplification of the pET-Duet-1 vector
  • Assessment of the safety requirements
  • Preparing stocks for antibiotics, glycerol, LB & MilliQ


Week 27

- The Clone Wars

Gibson Assembly:
  • Linearizing pET-Duet-1 for Gibson Assembly
  • Our first Gibson Assembly!

Traditional cloning:
  • Amplification of the pET-Duet-1 vector
  • Nde1 & Kpn1 digestion of the pET-Duet-1 vector (MCS-2) for traditional cloning


Week 28

- Et tu, pET-Duet?

Gibson Assembly:
  • Linearizing pET-Duet-1 for Gibson Assembly and debugging
  • Digestion of the template using DpnI
  • Running a gel to check whether the linearization was successful
  • Gibson Assembly for MCS1
  • Transformation and amplification of the plasmid

Traditional cloning:
  • Amplification of the inserts
  • Xbal & Pstl digestion of the pET-Duet-1 vector (MCS-1)
  • Xba1 & Pst1 digestion of the inserts (MCS-1)
  • Nde1 & Kpn1 digestion of the inserts (MCS-2)
  • Ligation
  • Transformation in NB
  • Colony PCR & gel electrophorese: The inserts are succesfully ligated
  • Culturing of the colonies with the correct plasmid
  • Making a glycerol stock & sending the DNA for sequencing


Week 29

- Hopeful results

Gibson Assembly:
  • Plasmid isolation, followed by sequencing of the insert on MCS1
  • Linearization of the vector on MCS2
  • Gibson Assembly of the second MCS
  • Colony PCR of MCS2, showing promising results!
  • Culturing and preparing for protein expression

Protein expression:
  • Double transformation of pET-Duet-1 (MCS1) and pEVOL in BL21.
  • Culturing & making a glycerol stock

Traditional cloning:
The sequencing results are positive, everything is built in correctly.


Week 30

- The moment of truth

Gibson Assembly:
  • Plasmid Isolation, followed by sequencing of the insert on MCS2

Protein expression of the plasmids containing MCS-1:
  • Culturing and protein expression of pET-Duet-1 (MCS-1)
  • Labelling the bacteria with DBCO-PEG4-Tamra
  • FACS results don't show the click reaction...

Double transformation & protein expression of the plasmids containing MCS-1 & MCS-2:
  • Double transformation, culturing and protein expression of pET-Duet-1 (MCS-1 & MCS-2)
  • The click reaction occured according to FACS results

Traditional cloning
  • The vector which already contained MCS-1 is digested at MCS-2
  • Ligation of vector & insert. This results in a plasmid containing MCS-1 (OmpX-NanoLuc) and MCS-2 (OmpX-neongreen)


Week 31

- busy times

FACS:
  • Protein expression of bacteria containing MCS-1 and bacteria containing MCS-1 & MCS-2
  • FACS (labelling bacteria with DBCO-PEG4-tamra) shows a click reaction with bacteria containing MCS-1 & MCS-2. No click reaction occurs with bacteria containing only MCS-1

Traditional Cloning:
  • Transform of the newly created plasmid MCS-1 (OmpX-NanoLuc) and MCS-2 (OmpX-neongreen)
  • Colony PCR

Gibson Assembly:
  • Succesfull double transformation of 12.2 at 20 ng/uL (rather than 4)
  • Gibson Assembly, Colony PCR and transformation of NeonGreen into MCS1


Week 32

-

FACS:
  • Protein expression of bacteria containing MCS-1 and bacteria containing MCS-1 with mNeongreen

Traditional Cloning:
  • Protein expression of constructs with mNeongreen and Nanoluc
  • Luminescence and fluorescence assays of expressed proteins
  • Verification of protein expression using a 10% SDS-PAGE gel

Gibson Assembly:
  • Succesfull double transformation of construct 14
  • Sequencing of the constructs