Team:TU Eindhoven/Notebook/Timeline

June

Wetlab

• Arrival of the biobricks
• The designs for the plasmids are approved and ordered Primers
• Preparation of culture media, agar plates, antibiotics and glycerol 
• Amplification of PET 29a(+), pEVOL pAzF, BBa_K811005(Penn) & CPX

July

Week 27: June 30 – July 6

Wetlab

• Plasmid amplification and culturing BBa_K811005(Penn) + CPX
• Vector and insert Digestions
  
• BBa_K811005(Penn) + CPX insert ligate pET29a 
  
• Colony PCR

Microfluidics lab

• Photo lithography
• Soft lithography
• Testing polyacrylamide device with dye
• Protocol aquapel
• New polyacrylamide devices: Photo lithography
• Improved protocol with shorter ashing time, aquapel coating improvement, shorter baking time PDMS.

Week 28: July 7 – July 13

Wetlab

• Colony PCR
• Site directed mutagenesis of CPX

Microfluidics lab

• Create oil and water phase, testing devices in ICMS lab
• Photo lithography (height measurement + silanization)
• Control polyacrylamide droplet with Leica microscope and with phantom V9
• Droplets separation (centrifuge) and view with microscope

Week 29: July 14 – July 20

Wetlab

• Site directed mutagenesis of CPX
• Sequencing of CPX and BBa_K811005(Penn)

Microfluidics lab

• Create oil and water phase
• Control polyacrylamide droplet with Leica microscope and with phantom V9
• Droplets separation (filtration) and view with microscope
• Soft lithography (no glass binding)
• Soft lithography (glass binding)
• Protocol aquapel
• Create oil and water phase
• Droplets separation (centrifuge) and view with microscope

Week 30: July 21 – July 27

Wetlab

• Sequencing results came in and were as expected
• Protein expression
• FACS – DBCO PEG4 5/6 TAMRA

Microfluidics lab

• Soft lithography (glass binding)
• Protocol aquapel
• Control polyacrylamide droplet with Leica microscope and with phantom V9
• Photo lithography (no height measurement + silenization)

Week 31: July  28 – August 3

Wetlab

• Protein expression
• FACS – DBCO PEG10kDa

Microfluidics lab

• Photo lithography (height measurement + silenization)
• Soft lithography (glass binding)
• Treat polyacrylamide and cell encapsulation devices with aquapel
• Control polyacrylamide droplet with Leica microscope and with phantom V9
• Droplets separation (centrifuge) and view with microscope

August

Week 32: August 4 – August 10

Wetlab

• Protein expression
• FACS – Antibody titration
• Protein expression curve

Microfluidics lab

• Control polyacrylamide droplet with Leica microscope and with phantom V9
• Protocol aquapel
• Droplets separation (centrifuge) and view with microscope
• Photo lithography
• Polyacrylamide droplets

Week 33: August 11 – August 17

Wetlab

• FACS DBCO PEG 5kDa fluorescent 
• Primers designed for biobricking
• Primers ordered for biobricking

Microfluidics lab

• Control polyacrylamide droplet with Leica microscope and with phantom V9
• Photo lithography (no aquapel and no glass binding)
• Droplets separation (centrifuge) and view with microscope
• Photo lithography (aquapel and glass binding)

Week 34: August 18 – August 24

Wetlab

• Side directed mutagenesis COMPx (BBa_K1492000) to mutate PSTI out of the sequence without changing the amino acid sequence
• Overhang PCR tRNA synthetase (BBa_K1492002) & COMPy (BBa_K1492001)
• Digestion PCR linearized backbone PSB1C3
• COMPx sent for sequencing
• Running agarose gel with tRNA synthetase and COMPy
• We did not see a crispy band at the lane of the tRNA synthetase but we did see a clear crispy band at the lane of COMPy.

Microfluidics lab

• Droplets separation (centrifuge) and view with microscope in ML II lab in Helix)
• Control polyacrylamide droplet with Leica microscope and with phantom V9
• Droplet separation by adding first HFE 7500 and then PFO to gain better control over the two phases
• Wafer production of cell encapsulation devices. After wafer was developed PDMS treatment was performed with 60 g PDMS instead of 50 g
• Cutting and ashing the new devices

Week 35: August 25 – August 31

Wetlab

• Ligase of PSB1C3 with COMPy
• Performed again overhang PCR for tRNA synthetase
• Sequencing results COMPx gave a negative result so we send another 5 samples for sequencing
• Running agarose gel with tRNA synthetase
• We did not see a crispy band at the lane of the tRNA synthetase again
• Performing colony PCR on the ligation product. The results showed an unsuccessful ligation
• Sequencing results gave another negative result

Microfluidics lab

• Treat devices with aquapel. After baking some debris was found in the devices
• Droplet formation with new devices to optimize the flow rates for bead encapsulation later. The flow rates were adjusted accordingly to W1:W2:O of 2:2:0.5
• Continuing optimizing flow rates for bead encapsulation. A different ratio of flow rates was used of W1:W2:O of 1:1:0.5

September

Week 36: September 1 – September 7

Wetlab

• Performed another Side directed mutagenesis COMPx  to mutate PSTI out of the sequence without changing the amino acid sequence
• Performed another colony PCR on the ligation product. The results showed an unsuccessful ligation again
• Checked the primers for the overhang of the tRNA synthetase and COMPy again. Due to a miscommunication wrong primers were designed we found out
• Designed new primers for COMPx, COMPy and the tRNA synthetase
• Ordered new linear backbone PSB1C3
• Synthesis and purification of DNA DBCO PEG4 product

Microfluidics lab

• PDMS treatment
• Fluorescent beads finally arrived. Cutting and ashing devices
• Aquapel treatment of the devices
• Encapsulation of beads
• Some devices seemed to be too stiff causing cracks in the PDMS which resulted in poor attachment of tubing
• Bead encapsulation. Stiff devices causing cracks resulting in loose tubing

Week 37: September 8 – September 14

Wetlab

• Sequencing result showed a successful mutation, however due to an error in the primer another mutation was introduced. However we chose to continue with this gene for overhang PCR and do another side directed mutagenesis at the end
• Primers and backbone we ordered arrived
• Overhang PCR of tRNA synthetase, COMPy and COMPx
• Digestion of linearized backbone
• Digestion of PCR products
• Reacting DNA DBCO with cells and testing reaction using FACS
• Creating circular DNA template for use in Rolling Circle Amplification
  Protein expression
• Cell viability test 1

Microfluidics lab

• PDMS pouring on the wafer. Bake PDMS for 1.15 h for more flexible devices
• Cut PDMS and ash devices
• Treat devices with Aquapel
• Could not run tests due to maintenance of the microscope
• Tests with bead encapsulation successful. Fluorescent pictures obtained however fluorescent signal intensity of single beads was too low to be detected

Week 38: September 15 – September 21

Wetlab

• We chose to culture the vector of a previously ordered biobrick we used for our project. (BBA_K811005) To make sure we had enough vector for our ligation
• Running agarose gel of tRNA synthetase, COMPy and COMPx. This time we saw nice crispy bands at the right place
• Mini prepping, digestion and gel extraction of the cultured vector PSB1C3
• Second attempt at creating circular template (previous product was impure)
• Testing of Rolling Circle and Multiple Chain Amplification without cells on agarose gel

Microfluidics lab

• Soft lithography of PDMS. Made two batches (= 10 devices)
• Cut PDMS and ash devices
• Aquapel devices. Two devices showed to have debris left after aquapeling
• Bead encapsulation. Beads coagulated in the channels
• Try washing used devices to remove beads. No movement observed. Beads still coagulated in other devices

Week 39: September 22 – September 28

Wetlab

• Ligation of tRNA synthetase, COMPy and COMPx into PSB1C3
• Colony picking of ligation product. For every insert we had a positive result
• Vectors sent for sequencing
• Testing of Rolling Circle and Multiple Chain Amplification without cells on agarose gel

Microfluidics lab

• Photo lithography
• No height measurement due to broken equipment in lab
• Soft lithography, PDMS treatment and baking
• Cut devices and ash. Three out of five devices were usable due to bad oxygen ashing
• Start small culture
• Treat devices with aquapel. No dirt seemed to be visible after drying with pressurized nitrogen
• Start protein expression. First OD measurement was 0.953. Continued inducing with IPTG
• After protein expression OD was 0.343 which equals 2.74*108 cells/mL
• Testing devices with fluorescent bacteria.

October

Week 40: September 29 – September 5

Wetlab

• Side directed mutagenesis to correct the mutated amino acid in the PSTI mutation
• Sequencing results were successful and positive
• Mutated vector send for sequencing
• Testing of Rolling Circle Amplification without cells on agarose gel
• Protein expression
• Cell viability test 2

Microfluidics lab

• Small culture
• Protein expression
• Testing devices with fluorescent bacteria. Bacteria were visible and devices were working. It was hard to stabilize the flow

Week 41: October 6 – October 12

Wetlab

• Sequencing results were successful and positive
• Biobrick sent on 8 october!
• Testing Rolling Circle Amplification on cells, verify using FACS
• Protein expression
• Cell viability test 3

Microfluidics lab

• Cut and ash devices. Ashing protocol adjusted to RF power: 30 W; vent time: 60 s; bleed time: 20 s.W at a pressure of 0.9 mbar
• Start small culture
• Treated devices with aquapel. One device appeared to show no binding with the glass. Start protein expression
• OD after protein expression was 0.94. This equals to 1.5*1010 cells/mL in the stock solution. Due to maintenance of the microscope bacteria were stored till the next day
• Droplet encapsulation was tested with polyacrylamide devices. Droplets were formed successfully. A new oil phase was made due to breaking of the droplets to ensure the quality of the oil phase. After this the problem was solved.

Week 42: October 13 – October 19

Microfluidics lab

• Start small culture.
• Start protein expression.
• Droplet encapsulation was tested with polyacrylamide devices and fluorescence bacteria