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Revision as of 17:18, 18 September 2015





Timeline



Somewhere in the beginning of July, we started our wetwork. We started the summer off working in smaller teams: we had a team working on Gibson Assembly, with traditional cloning and a team working on Interlab. In this way, we could fit tons of wetwork within the remainder of the summer. Below, we hope to give a short overview of how we started off and which experiments we carried out in which weeks.



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: sequencing results came back disastrous. It seemed as if pETDuet-1 had turned on us. In the end, however, it turned out that we had used the wrong primers. Always use the right primers, folks!
  • 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

- shine some light

FACS:
  • Protein expression of bacteria containing a split luciferase in MCS-1 and bacteria containing mNeongreen in MCS1

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

Gibson Assembly:
  • Succesfull double transformation of pEvol plasmid & plasmid containing Neongreen in MCS1.
  • Sequencing of the constructs


Week 33

- Oh when it all, it all falls down

Gibson:
  • Linearization of the pETDuet-1 vector containing the NeonGreen insert
  • Gibson Assembly of the linearized vector with the NanoLuc insert
  • Linearization of pETDuet-1 at MCS1
  • Gibson Assembly of the linearized pETDuet-1 vector with NanoLuc
  • Miniprepping and double transformation of the NL-vectors

FACS:
  • Protein expression of constructs with mNeongreen and Nanoluc
  • Luminescence and fluorescence assays of expressed proteins: neongreen is present
  • Verification of protein expression using a 10% SDS-PAGE gel
  • Setting up a crime scene

  • Succesful double transformation of pEvol plasmid & plasmid containing Neongreen in MCS1.
  • Sequencing of the constructs


Week 34

- Sherlocking our way to salvation

FACS:
  • Protein expression of all NG+NL containing vectors
  • Redoing some transformations
  • Measuring bioluminescence & fluorescence
  • FACS'ing all expressed constructs
  • Salvation: a faulty transformation had caused trouble
    • Gibson:
  • Miniprepping and analyzing some constructs through PCR
  • Replacing NanoLuc with mTurquoise to obtain a FRET-sensor as a back up plan


Week 35

- How did it get so late so soon?

Gibson:
  • Finalizing the backup construct with mTurquoise
  • Gibson Assembly of SmBiT in MCS2
    Protein Expression:
    • Obtaining all data about bioluminescense and fluorescense
    • Testing the sensor with complementarty DNA
    • Testing aptamers + thrombin