Team:TU Eindhoven/Notebook





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, a team responsible for Traditional Cloning and a team working on Interlab. In this way, we could fit tons of wetwork within the few weeks of the summer holidays. Below, we hope to give a short overview of how we started off and which experiments we carried out in which weeks.

See the black timeline below for a global impression of the major experiments carried out by the teams working with Gibson Assembly and Traditional cloning. These experiments brougth us were we are today and results can be seen here.



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 MCS-1 of pET-Duet-1 for Gibson Assembly
  • Digestion of the template using DpnI
  • Running a gel to check whether the linearization was successful
  • Our first Gibson Assembly!
  • Plasmid amplification into NEB 5-alpha

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 MCS-1 of 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 MCS-1
  • Plasmid amplification into NEB 5-alpha

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 MCS-1
  • Linearization of the vector on MCS-2
  • Gibson Assembly of MCS-2
  • Plasmid amplification into NEB 5-alpha
  • Colony PCR of MCS-2, 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 MCS-2

Protein expression of the plasmids containing MCS-1:
  • Culturing and protein expression of MCS-1 containing pET-Duet-1 vector
  • 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

Gibson Assembly:
  • Succesfull double transformation of 12.2 at 20 ng/uL (rather than 4)
  • Gibson Assembly, Colony PCR and transformation of mNeonGreen into MCS-1

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


Week 32

- Shine some light

Gibson Assembly:
  • Succesfull double transformation of pEVOL & pET-Duet-1 containing mNeonGreen in MCS-1 in BL21(DE3).
  • Sequencing of the constructs

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

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


Week 33

- Oh when it all, it all falls down

Gibson:
  • Linearization of the pETDuet-1 vector containing the mNeonGreen insert
  • Digestion of the template using DpnI
  • Running a gel to check whether the linearization was successful
  • Gibson Assembly of the linearized vector with the NanoLuc insert
  • Double transformation into BL21(DE3) and colony PCR to check whether NanoLuc is correctly inserted
  • Linearization of pETDuet-1 at MCS-2
  • Digestion of the template using DpnI
  • Running a gel to check whether the linearization was successful
  • Gibson Assembly of the MCS-2 linearized pETDuet-1 vector with NanoLuc
  • Double transformation into BL21(DE3) and colony PCR to check whether NanoLuc is correctly inserted
  • Small culturing, miniprepping and double transformation of the NanoLuc-containing vectors in BL21(DE3)

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


Week 34

- Sherlocking our way to salvation

Gibson Assembly:
  • Troubleshooting of construct 8.2.
  • Miniprepping and analyzing some constructs through PCR
  • Replacing NanoLuc with mTurquoise2 to obtain a FRET-sensor as a back up plan, which included linearization, ligation and transformation

FACS:
  • Protein expression of all NG+NL containing pET-Duet-1 vectors
  • Redoing some transformations
  • Measuring bioluminescence & fluorescence
  • FACS'ing all expressed constructs
  • Salvation: a faulty transformation had caused trouble


Week 35

- How did it get so late so soon?

Gibson:
  • Finalizing the backup construct with mTurquoise2

Protein Expression:
  • Obtaining all data about bioluminescence and fluorescence


Week 36

- Quantification time!

Protein Expression:
  • Obtaining more data about bioluminescence and fluorescence
  • Testing the sensor with complementary DNA


Week 37

- Extensive testing

Protein Expression:
  • Characterization of the binding of complementary DNA strands labeled with a fluorescent dye to DBCO-TEG4-DNA strands
  • Characterization of mNeonGreen and of mTurquoise2
  • Troubleshooting the complementary DNA

Alginate beads:
  • Preparing of the alginate solution and buffers
  • Making of the first alginate beads with empty cells and with pure GFP


Week 38

- The final countdown...

FACS:
  • FACS'ing the fluorescent-labeled complementary DNA strands

Protein Expression:
  • Characterization of the binding of complementary DNA strands labeled with a fluorescent dye to DBCO-TEG4-DNA strands
  • Repeated characterization of mNeonGreen and of mTurquoise2

Alginate beads:
  • Making of the alginate beads with GFP producing cells