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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. | 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. | ||
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Revision as of 12:17, 18 September 2015
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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:
Traditional cloning:
- 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:
Traditional cloning:
- 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:
Protein expression:
Traditional cloning:
The sequencing results are positive, everything is built in correctly.
- 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:
Protein expression of the plasmids containing MCS-1:
Double transformation & protein expression of the plasmids containing MCS-1 & MCS-2:
Traditional cloning
- 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:
Traditional Cloning:
Gibson Assembly:
- 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:
Traditional Cloning:
Gibson Assembly:
- 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:
FACS:
- 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:
Protein Expression:
- Obtaining all data about bioluminescense and fluorescense
- Testing the sensor with complementarty DNA
- Testing aptamers + thrombin