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Notebook Introduction:

Here we document the timeline of our project. See the project ideation page for further details regarding the early months of the project. Furthermore, the modelling, education and outreach and human practice aspects of the project were ongoing throughout the project and ran parallel to our efforts in the wet-lab. This notebook aims to document the milestones associated with our wet-lab work. In brief:

July:
Finalised project idea - Robust, Antithetic Integral Controller!
Met virtually with Dr Corentin Briat and Dr Stephanie Aoki
Got our lab memberships (Thanks Biomakespace!)
Lab safety inductions and further lab-safety considerations
Part mining sessions underway

August:
3D Print Contribution
[Main project] Cloning lvl 0s
[Main project] Cloning lvl 1s

September:
JUMP DV Characterisation Experiment
P_BAD Characterisation Experiment
StayGold Characterisation Experiment
[Main project] Cloning level 2s

We have documented our lab work starting from the week we obtained our distribution kit (as this was necessary for us to begin work). This was the third week of August and so we were in the lab for approximately 7 weeks in total.

Lab Week 1:

• Finally got our hands on a distribution kit!! Much later than we hoped.


• PCRed CDS: ECF16, ECF20, SinI, SinR, AS16, TetRDN, AraE, pBADmin, PSigW, pSinR, p20, p16, RSiW, VanR, AS20, StayGold, SigW, DT54, DG352125, MegaT, DT5, DT3 L3S2 full device


• Repeat PCR on DNA parts that have low yield from previous PCRs
  p_SinR, P_SigW, DT5, L3S2P55 Terminator, DT3, P_20, ECF16, Full device

Lab Week 2:

• Attempt interlab - but decide to spend our time focused on our main project


• JUMP LVL0 cloning


• PCR of 7A, 7M, 9C, 9K, 9O,10G, 13A,13C, 16K, 22A, 22I from dist. Kit. 1 rePCRed 4C and 4E


• cPCR (AS16, p16, pSigW)
  Realised its not AS16 but SinR! We made a mistake during order placement - ordered both as a sinR sequence!!


• GGA of the remaining 20 ordered DNA into Level 0:
  AraE, AS20, ECF16, ECF20, RSiW, SigW, sinI, p_sinR, StayGold, TetRDN, vanR, p20, p_badmin,sinR, T_DT3, T_DT5, T_DT54, T_full device, T_terminator module, T_megaT


• Miniprep and Chemical transformation of GGA (20 remaining fragments)


• cPCR of the remaining 20


• Mini prep of AraE, AS20, ECF16, ECF20, RsiW, SigW, SinI, SinR. Stay Gold, TetR^DN, VanR, P_20, P_badmin, P_SinR, T_DT3, T_DT5, T_DT54, T_Full, T_Term, T_Mega


• Chemical transformation of DK Plate 2 GFPs (afraGFP, efasGFP, eechGFP1)


• Failed to obtain tetR and Marionette strain cells
  tetR can be obtained from DK but we need it for linear GGA decided we will order an adapter to allow us to use the tetR part from DK
  Will try to find Marionette strain as needed for our characterisation experiment Obtained CIDAR MoClo (Amp):
  Anderson promoter: J2323102, J23106, J23116
  RBS: B0032m, B0033m, B0034m
  Promoter: pBAD


• Miniprep Marionette cells and the 3 GFPs


• Molaritised Level 0 vectors with our parts and Level 1 acceptor vectors level 1A and 1C acceptor, p_cinR, p_vanCC, P_20992, P_sinR, B0032, B0034, AS20, SinI, cinR^AM, vanR^AM, DT5, MegaT


• GGA Level 1 assembly trial run


• Chemical transformation of the GGA product


• Start Characterising JUMP DV experiment: Transforming 2M(pBBRI low-med copy no. pJUMP23-1A) and 4I (pUC high copy no. pJUMP28-1A) from DK Plate 1

Week 3:

Lvl0 GGA of pSinR and DT3 and ran gel
Sequenced lvl 1 10, 25, 26, 27, 28, 29, 30, 31, 32, 33, 36

10✔: (characterisation) Mod A - p_20992, B0032m, sgGFP, DT5
25✔: (Perturbation) Mod D - P_Tet*, B0032, CinR^AM, MegaT
26✔: (Perturbation) Mod D - P_Tet*, B0032, VanR^AM, MegaT
27: (global) Mod A - P_16_3622, B0032, CinR^AM, MegaT (megaT mutated!)
28: (global) Mod A - P_16_3622, B0034, CinR^AM, MegaT (megaT mutated!)
29: (global) Mod B - J23106, B0034, ECF16, DT3 (needs to be redone)
30: (global) Mod B - J23105, B0034, ECF16, DT3 (med. Transform. success)(needs to be redone)
31: (global) Mod B - J23100, B0032, ECF16, DT3 (needs to be redone)
32: (global) Mod B - J23106, B0032, ECF16, DT3 (needs to be redone)
33✔: (global) Mod B - J23105, B0032, ECF16, DT3
36✔: (characterisation) Mod A - p_163622, B0032m, sgGFP, DT5

Plated one of our only lvl1s for JUMP DV characterisation experiment from glycerol stock

cPCR and gel of 15, 16, 17, 24, pSinR1, pSinR2 and DT3

Sequenced A, B, C, D and 27 FP and 28 FP

JUMP DV CHARACTERISATION:
Innoculated liquid culture of ‘Just cells’ (neg. control), ‘pSC101’ (low copy no.), ‘pBBR1’ (low-med. copy no.), ‘pUC’ (high copy no.), ‘lvl1’ (neg. Control, no sfgfp)

Nanodropped samples and 44 (pbad)

Sequenced pSInR1, pSinR2, DT3, 17, 24.1, 24.3, 44fw, 44rv

GGA

cPCR

JUMP DV CHARACTERISATION: Test 1 using ammonium sulphate concentration 0-1000mM

Gel of cPCRed lvl 1s 1, 3, 4, 5, 6, 7, 8, 9, 11, 15, 18, 22, 23, 37, 39.

1: (global) Mod A: P_20_992, B0032, CinR^AM, MegaT (med. Transform. success)
3: (global) Mod B: J23106, B0034, ECF20, DT3 (med. Transform. success)
4: (global) Mod B: J23105, B0034, ECF20, DT3 (transform. unsuccessful)
5: (global) Mod B: J23100, B0032, ECF20, DT3 (med. Transform. success)
6: (global) Mod B: J23106, B0032, ECF20, DT3 (transform. unsuccessful)
7✔: (global) Mod B: J23105, B0032, ECF20, DT3
8: (global) Mod C: P_Cin, B0032, AS20, DT5
9: (global) Mod C: P_Cin, B0033, AS20, DT5 (transform. unsuccessful)
11: (characterisation) Mod B: P_bad, B0032m, ECF20, DT3
15: (global) Mod B: J23100, B0032, SinR, DT3
18: (global) Mod B: J23106, B0033, SinR, DT3
22: (characterisation) Mod B: P_bad, B0032m, SinR, DT3 (no colonies on transformed plate)
23: (characterisation) Mod C: P_tet*, B0032m, SinI, DT54
37: (characterisation) Mod B: P_bad, B0032m, ECF16, DT3
39: (negative feedback) 1A - pJUMP27: P_vanCC, B0032m, vanR^AM, MegaT

Sequenced: pbad, ptet*, tetR, J23106, level 1 (1), 3, 4, 5, 6, 7, 8, 9, 18, 22, 23, 1.

JUMP DV CHARACTERISATION: Collected results of test 1 from plate reader and inoculated liquid cultures for test 2

Week 4

• cPCR


• Nanodropped level 1: 41, B0032, level 1 9.2, level 1 22, level 1 (1), pbadwt


• Sequenced: level 1s: 22, 41, 3.2, 4.2


• GGA of 5, 6, 25, 26, 45, 46:
  5: (global) Mod B: J23100, B0032, ECF20, DT3
  6: (global) Mod B: J23106, B0032, ECF20, DT3
  25: (Perturbation) Mod D - P_Tet*, B0032, CinR^AM, MegaT
  26: (Perturbation) Mod D - P_Tet*, B0032, VanR^AM, MegaT
  45: (StayGold characterization) 1A - pJUMP27: P_tet*, B0032m, afraGFP, DT5.
  46: (StayGold characterization) 1A - pJUMP27: P_tet*, B0032m, efasGFP, DT5.


• Ran gel of 12, 13 and 40


• JUMP DV CHARACTERISATION: ran test 2 with ammonium sulphate concentration 0-720mM


• Nanodropped samples: 12, 13, 40 and 42.


• Sequenced level 1s: 12, 13, 40, 42, 22


• GGA of samples 1, 3, 25, 49:
  1: (Global) Mod A: P_20_992, B0032, CinR^AM, MegaT
  3: (Global) Mod B: J23106, B0034, ECF20, DT3
  25: (Perturbation) Mod D - P_Tet*, B0032, CinR^AM, MegaT
  49: (Open loop perturbation) Mod D - J23106, B0032m, tetR, DT54.


• Ran gel of samples: 5, 6, 45 and 46


• Overnight GGA of: 11,12, 39, 48:
  11: (characterisation) Mod B: P_bad, B0032m, ECF20, DT3
  12: (characterisation) Mod C: P_tet*, B0032m, AS20, DT54
  39: (Negative feedback): 1A - pJUMP27- P_vanCC, B0032m, vanR^AM, MegaT
  48: (Open loop perturbation): Mod C - P_tet*, B0032m, ECF20, DT5.


• Sequenced samples: ECK12009600, J23106 (old), J23106 (new), J23100 (13C-2), J23105 (13A-1), Level 1 5, Level 1 45.2, Level 1 46.2


• Nanodropped samples: Level 1 5, level 1 45.1, level 1 45.2, level 1 45.3, level 1 46.1, level 1 46.2, level 1 46.3, J23102, J23106, 8E.


• GGA:
  16: (Global): Mod B - J23106, B0032, SinR, DT3
  20: (Global): Mod C - P_VanCC, B0032, SinI, DT5
  50: (Open loop perturbation): Mod C, P_tet*, B0032m, sinR, DT5
  JUMP27 DSS
  8E DSS


• Miniprep of samples 1, 3 and 25:
  1: (Global) Mod A: P_20_992, B0032, CinR^AM, MegaT
  3: (Global) Mod B: J23106, B0034, ECF20, DT3
  25: (Perturbation) Mod D - P_Tet*, B0032, CinR^AM, MegaT


• Sequenced: 1.2 (fp - forward primer), 1.2 (rp - reverse primer), 3.1 (rp), 25.3 (fp), 25.3 (rp)

Week 5:

• Nanodropped lvl 0 samples ‘MegaT with RFP + SpaI’ and AS16


• GGA of 27, 28, 3?, 51, 34, 35, 38, 3, 11, 48, 16:
  3: (Global): Mod B - J23106, B0034, ECF20, DT3
  11: (characterisation) Mod B: P_bad, B0032m, ECF20, DT3
  16: (Global): Mod B - J23106, B0032, SinR, DT3
  27: (global): Mod A - P_16_3622, B0032, CinR^AM, MegaT
  28: (global): Mod A - P_16_3622, B0034, CinR^AM, MegaT
  34: (Global): Mod C - P_Cin, B0032, AS16, DT5
  35: (Global): Mod C - P_Cin, B0033, AS16, DT5
  38: (Characterisation): Mod C - P_tet*, B0032m, AS16, DT54
  48: (Open loop perturbation): Mod C: P_tet*, B0032m, ECF20, DT5


• Sequenced 39.1 (fp), 39.1 (rp), 1.2 (rp)


• cPCR of 26, 43, 13, 21, 16, 20, 50, Jump27 DSS, 3, 49


• Lvl 0 GGA - pBAD experiment MegaT, AS16, MegaT RFP, KanR, SCFP34, Pbadwt, Pbadneg, PbadAP1, Pbad AP2, PbadAP3, PbadAP4, PbadAP5, PbadAP6, PbadAP7, PbadAP8, Pbadmin.


• Gel of 26, 43, 13, 21, 20, 50, 49, DSS


• Sequenced 26 (fp), 26 (rp), 43 (rp), 13 (fp), 13 (rp), 21 (rp), 20 (rp), 50 (rp), 3 (rp), 49 (rp), 39 (fp).


• Nanodropped 20.2, 20.3, 13.1, 13.2, 3.3, 50.1, 43.1, 43.2, 49.1, 49.2, 26.1, 26.2, 21.1, 21.3


• Gel of 27, 28, 34, 35, 37, 38, 51, pJUMP, lvl2


• GGA of 11, 48, 16:
  11: (characterisation) Mod B: P_bad, B0032m, ECF20, DT3
  14: (Global): Mod A- P_SinR, B0034, VanR^AM, MegaT (Moved onto repressive?)
  16: (Global): Mod B - J23106, B0032, SinR, DT3


• Nanodropped 20, 21, 22, 23, 24


• GGA:
  13: (Global): Mod A- P_SinR, B0032, VanR^AM, MegaT
  20: (Global): Mod C - P_VanCC, B0032, SinI, DT5
  49: (Open loop perturbation): Mod C - P_tet*, B0032m, ECF20, DT5
  50: (Open loop perturbation): Mod C, P_tet*, B0032m, sinR, DT5
  27: (global): Mod A - P_16_3622, B0032, CinR^AM, MegaT
  28: (global): Mod A - P_16_3622, B0034, CinR^AM, MegaT
  Lvl2 SinR: pJump47 (8E), 21, 22, 23, 24


• Sequenced 34, 35, 37, 38, 51, pBAD4, pBAD5, pBAD10


• Nanodropped 34, 30, 35, 37, 38, 51


• GGA (all have mVenus instead of sfGFP):
  10: (characterisation) Mod A - p_20992, B0032m, mVenus, DT5
  21: (Characterisation): Mod A - p_sinR, B0032m, mVenus, DT5
  36: (characterisation) Mod A - p_163622, B0032m, mVenus, DT5
  mV1: Mod A - J23100, B0032, mVenus, DT5


• GGA Anthony’s p_BAD experiment:
  PB2.8 - pJUMP27: pBADAP8, B0032, mVenus, DT5
  PB2.9 - pJUMP27: pBADmin, B0032, mVenus, DT5
  PB2.10 - pJUMP27: pBADwt, B0032, mVenus, DT5


• Transformation of above


• PCR on 2x mVenus DNA fragments - we were unsure if our last attempt to add the JUMP fusion sites to mVenus had worked


• Ran gel - they were the right size!


• Ran Gel of 10, 13, 20, 21, 27, 28, 29, 36, 48, 49 and Sin.
  10: (characterisation) Mod A - p_20992, B0032m, mVenus, DT5
  13: (Global): Mod A- P_SinR, B0032, VanR^AM, MegaT
  20: (Global): Mod C - P_VanCC, B0032, SinI, DT5
  27: (global): Mod A - P_16_3622, B0032, CinR^AM, MegaT
  28: (global): Mod A - P_16_3622, B0034, CinR^AM, MegaT
  29: (global) Mod B - J23106, B0034, ECF16, DT3
  36: (characterisation) Mod A - p_163622, B0032m, mVenus, DT5
  48: (Open loop perturbation): Mod C - P_tet*, B0032m, ECF20, DT5
  49: (Open loop perturbation): Mod C - P_tet*, B0032m, ECF20, DT5
  Lvl2 SinR: pJump47 (8E), 21, 22, 23, 24


• Same day GGA of 4 pBAD lvl1s and 4 lvl1s from our main project PB2.1, PB2.2, PB2.3, PB2.4, 48, 49, 50, 51:
  PB2.1 - pJUMP27: pBADAP1, B0032, mVenus, DT5
  PB2.2 - pJUMP27: pBADAP2, B0032, mVenus, DT5
  PB2.3 - pJUMP27: pBADAP3, B0032, mVenus, DT5
  PB2.4 - pJUMP27, pBADAP4, B0032, mVenus, DT5
  48: (Open loop perturbation): Mod C - P_tet*, B0032m, ECF20, DT5
  49: (Open loop perturbation): Mod C - P_tet*, B0032m, ECF20, DT5
  50: (Open loop perturbation): Mod C, P_tet*, B0032m, sinR, DT5
  51: (Open loop perturbation): Mod C, P_tet*, B0032m, ECF16, DT5


• Transformed the cells



















Week 6:

• cPCR 13 and 28:
  13: (Global): Mod A- P_SinR, B0032, VanR^AM, MegaT
  28: (global): Mod A - P_16_3622, B0034, CinR^AM, MegaT


• cPCR of 48, 49, 51 pB2.1, pB2.2, pB2.3, pB2.4


• cPCR of 2, 11, 14, 15, 18, 19, 21, 31, 32, 36, AS16 (lvl0), mVenus(lvl0) and MegaT (lvl0):
  2: (global) Mod A- P_20_992, B0034, CinR^AM, MegaT
  11: (characterisation) Mod B: P_bad, B0032m, ECF20, DT3
  14: (Global): Mod A- P_SinR, B0034, VanR^AM, MegaT
  15: (global) Mod B: J23100, B0032, SinR, DT3
  18: (global) Mod B: J23106, B0033, SinR, DT3
  19: (global) Mod C: P_VanCC, B0034, SinI, DT5
  21: (Characterisation): Mod A - p_sinR, B0032m, mVenus, DT5
  31: (global) Mod B - J23100, B0032, ECF16, DT3
  32: (global) Mod B - J23106, B0032, ECF16, DT3
  36: (characterisation) Mod A - p_163622, B0032m, mVenus, DT5


• Ran gel - 2.1, 2.2, 2.3, 11.3, 19.2, AS16 1, AS16 2, mV1, mV2, MT1, MT2 worked


• Miniprepped and Nanodropped the samples for sequencing


• cPCR of PB2.2, PB2.5, PB2.8, PB2.9, 21, 13, 14:
  13: (Global): Mod A- P_SinR, B0032, VanR^AM, MegaT
  14: (Global): Mod A- P_SinR, B0034, VanR^AM, MegaT
  21: (Characterisation): Mod A - p_sinR, B0032m, mVenus, DT5


• Ran gel - only one sample of PB2.9 was successful out of all of them.


• Miniprepped lvl2 1.1 and lvl0 MT RFP with new Monarch kit - PINK!


• Nanodropped the samples


• LEVEL 2! GGA of 5 Level 2s: 5, 8, 9, 10, 11:
  5: A Mod: P_BADmin, B0032, AS16, DT5 -B Mod: J23105, B0034, ECF16, DT54 -C Mod: P_16_3622, B0032, araC^AM, B0032 (m), mVenus, DT3 -D Mod: J23105, B0034, AraE, L3S2P55.
  8: A Mod: P_BADmin, B0032, ECF16, DT5 -B Mod: J23117, B0034, AS16, DT54 -C Mod: P_16_3622, B0034, araC^AM, B0032 (m), mVenus, DT3 -D Mod: J23105, B0034, AraE, L3S2P55.
  9: A Mod: P_BADmin, B0032, ECF16, DT5 -B Mod: J23117, B0034, AS16, DT54 -C Mod: P_16_3622, B0032, araC^AM, B0032 (m), mVenus, DT3 -D Mod: J23105, B0034, AraE, L3S2P55.
  10: A Mod: P_BADmin, B0032, ECF16, DT5 -B Mod: J23117, B0034, AS16, DT54 -C Mod: P_16_3622, B0033, araC^AM, B0032 (m), mVenus, DT3 -D Mod: J23105, B0034, AraE, L3S2P55.
  11: A Mod: P_BADmin, B0033, ECF16, DT5 -B Mod: J23100, B0032, AS16, DT54 -C Mod: P_16_3622, B0034, araC^AM, B0032 (m), mVenus, DT3 -D Mod: J23105, B0034, AraE, L3S2P55.


• cPCR of the samples


• Ran Gel - none worked :(