iGEM 2022 — Team cambridge

[Introduction](#intro)

[Week 1](#week1)

[Week 2](#week2)

[Week 3](#week3)

[Week 4](#week4)

[Week 5](#week5)

[Week 6](#week6)

Notebook

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# Notebook Introduction:

Here we document the timeline of our project. See the [project ideation](https://2022.igem.wiki/cambridge/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

![Time Line in Breif](https://static.igem.wiki/teams/4491/wiki/notebook/untitled-presentation-9.png)

Project Timeline in Brief

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

![](https://static.igem.wiki/teams/4491/wiki/notebook/week1.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week1-2.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week1-3.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week1-4.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week1-5.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week1-6.jpg)

# 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

![](https://static.igem.wiki/teams/4491/wiki/notebook/week2.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week2-2.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week2-3.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week2-4.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week2-5.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week2-6.jpg)

# 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

![](https://static.igem.wiki/teams/4491/wiki/notebook/week3.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week3-2.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week3-3.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week3-4.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week3-5.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/cells-1.jpg)

# 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)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week4.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week4-2.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week4-3.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week4-4.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week4-5.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/charlie.jpg)

# 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

![](https://static.igem.wiki/teams/4491/wiki/notebook/week5.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week5-2.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week5-3.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week5-4.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week5-5.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/cells-3.jpg)

# 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 :(

![](https://static.igem.wiki/teams/4491/wiki/notebook/week6.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week6-2.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week6-3.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/week6-4.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/joni-7.jpg)

![](https://static.igem.wiki/teams/4491/wiki/notebook/shauriandkaz-9.jpg)

{% endblock %}

[Summary](#summary)

Design of MegaT ▼

[Purpose](#MegaT)

[Design (cycle 1)](#Design1_mt)

[Build (cycle 1)](#Build1_mt)

[Test (cycle 1)](#Test1_mt)

[Learn (cycle 1)](#Learn1_mt)

[Design (cycle 2)](#Design2_mt)

Negative Autoregulation ▼

[Purpose](#neg)

[Design (cycle 1)](#Design1_neg)

[Build (cycle 1)](#Build1_neg)

[Test (cycle 1)](#Test1_neg)

[Learn (cycle 1)](#Learn1_neg)

[Design (cycle 2)](#Design2_neg)

Downstream site integration ▼

[Purpose](#DSS)

[Design (cycle 1)](#Design1_dss)

[Build (cycle 1)](#Build1_dss)

[Test (cycle 1)](#Test1_dss)

[Learn (cycle 1)](#Learn1_dss)

[Design (cycle 2)](#Design2_dss)

[References](#ref)

Engineering Success

---

# Summary

The process of Engineering Success requires the iteration through the Design-Build-Test-Learn Cycle. In this page, we present 3 examples of the team's Engineering Success

1. Design of the operon MegaT to overcome design constraints of our project

* **Design** RBS, reporter and terminator together as a part
* **Build** MegaT into a negative feedback circuit
* **Test** the effect of MegaT through microscopy
* **Learn** the results from the microscopy
* **Redesign** a family of MegaT suitable for other circuits

2. Design and Characterisation of an autoregulatory negative feedback circuit

* **Design** an autoregulatory negative feedback circuit
* **Build** the circuit with JUMP Golden Gate Assembly
* **Test** the circuit in the Mother Machine
* **Learn** the results from the Mother Machine Experiment
* **Redesign** the autoregulatory negative feedback loop to improve system dynamics

3. Insertion of the perturbation circuit into the downstream site of a JUMP acceptor vector
   * **Design** adapters to allow insertion into downstream site of JUMP vectors
   * **Build** the linear transcription unit with linear Golden Gate Assembly Pipeline
   * **Test** through colony PCR
   * **Learn** the result of successful integration of downstream site through sequencing
   * **Redesign** the transcription unit allowing easier selection for the next iteration

These Engineering processes are important in the process of our main project of creating an antithetic integral controller. The design of the operon MegaT allows us to measure the amount of output we have, thus showing adaptation. The negative feedback circuit as an example, utilises the operon MegaT to visualise the amount of output and serve as a comparison to the antithetic integral controller. To allow us to perturb the antithetic integral controller, we require both MegaT, showing the increase of production rate of output through perturbation and a downstream site that turns the perturbation on and off. The success of the perturbation circuit is determined by the design of the transcription unit that integrates into the downstream site of the Level 2 pJUMP47-2A acceptor.

# Design of MegaT

In our project, we hope to use a reporter protein to visualise the amount of feedback species X we have in the circuit. To avoid tagging the reporter protein directly onto X, we have decided to put in an RBS and a reporter (mVenus) right after X to visualise the amount of X. We are implementing JUMP assembly. However, JUMP assembly requires us to clone in 4 parts (usually the promoter, RBS, CDS and terminator) at the same time and does not allow polycistronic assemblies as is the case we have in our main project. Therefore, we need a way to design an operon to allow polycistronic assembly. For more details on JUMP Golden Gate Assembly, please refer to the [Rationale](https://2022.igem.wiki/cambridge/design#jump) page.

## Design (cycle 1)

To allow the reporter to be expressed under the same promoter as X, we have designed MegaT such that the RBS, reporter and terminator are attached together as a fragment. The MegaT part we have designed consists of RBS B0032, CDS mVenus and L3S2P21 terminator (Chen *et al.*., 2013) and is placed as an order with IDT.

We have chosen to use B0032 because we want to start with a medium strength of RBS. If the RBS is too strong, the reporter may form aggregates when existing in high amounts. If the RBS is too weak however, the amount of mVenus produced may not be detectable. In terms of the terminator, the L3S2P21 is a synthetic high efficiency terminator. A strong terminator is important to terminate transcription.

![Figure 3.](https://static.igem.wiki/teams/4491/wiki/engineering/megat-design-new1.png)

Figure 1. Design of the operon MegaT and how it fits into JUMP Golden Gate Assembly

## Build (cycle 1)

The MegaT part itself will not be expressing the reporter itself without the promoter part before it. Therefore, we have built a genetic circuit with a single transcription unit and use MegaT to visualise the relative amount of CDS we have. The circuit we have decided to characterise MegaT in is the autoregulatory negative feedback circuit (nFc). The purpose of this circuit is to characterise the behaviour of autoregulatory feedback circuit and compare it with other adaptation strategies. For more details please look at the part on [negative feedback](https://2022.igem.wiki/cambridge/engineering#neg) in this page.

## Test (cycle 1)

To test for the intensity of the reporter protein in the circuit and show that it is reporting the relative amount of VanR, we have cultured the cells with the plasmid containing the plasmid in 0µM and 100µM Vanillic acid overnight.
The following table shows the strain of cells and culture conditions overnight we have used for the characterisation of MegaT. The strain SB7 is a strain with mCherry integrated into chromosomal DNA.

| Strain | Plasmid | Medium (Overnight culture) | Antibiotics | Volume | Purpose |
| --- | --- | --- | --- | --- | --- |
| *E. coli* (SB7) | nFc | EZRDM - 0.1% pluronic | Kanamycin | 2mL | Test strain |
| *E. coli* (SB7) | nFc | EZRDM - 0.1% pluronic - 100µM Vanillic Acid | Kanamycin | 2mL | Test strain |
| *E. coli* (SB7) | No plasmid | EZRDM - 0.1% pluronic | Kanamycin | 2mL | Negative control |
Table 2. Overnight culture for characterisation of MegaT

| Channel | Reporter | Purpose |
| --- | --- | --- |
| phase contrast | NA | show the presence of cells |
| mCherry | mCherry | Show the presence of SB7 - all SB7 cells have mCherry integrated into their chromosome  For cell segmentation |
| YFP | mVenus | To show the concentration of the reporter |
Table 1. Microscope settings for the characterisation of MegaT

We are using the Nikon TI2 Eclipse microscope and have taken 9 images of each cell strain at different fields of views separated from each other to prevent imaging the same cell twice.

## Learn (cycle 1)

**Processing the image data**

**Importing images**

The microscope images we took came in .nd2 file where the images of separate channels are extracted into .tiff files using NIS Element Viewer. The tiff files are then imported into Python using the scikit image and pillow library and being converted into a 2D NumPy array.

**Cell Segmentation**

In image analysis, regions where cells are present will have a different value to the background. When analysing the images taken with fluorescence, the region where cells are present will be at a higher intensity value compared to the background. As all the cells we have imaged is the SB7 strain that has mCherry integrated into their genome, we make use of the mCherry images to segment cells.

To segment cells, we use the Omnipose library in Python, putting in the mCherry fluorescent images, to create masks in .png format to segment individual cells into separate regions.

![Figure 2.](https://static.igem.wiki/teams/4491/wiki/engineering/omnipose.png)

Figure 2. Cell segmentation of one of the field of views

**Finding properties of individual cells**

After segmentation, the mask has to be labelled i.e. to give each region of interest an associated label and the property of each labelled region can be found. This is done by the function label() from the scikit-image package in Python. After labelling the image, we can find the properties of individual regions which are then exported as Pandas DataFrame with the function regionprops\_table, also from the scikit-image package. In this context, area, mean intensity and major axis length are the properties that we are interested in.

![Figure 3.](https://static.igem.wiki/teams/4491/wiki/engineering/label-image.png)

Figure 3. The label image from one of the field of views

![Figure 4.](https://static.igem.wiki/teams/4491/wiki/engineering/regionprops-table.png)

Figure 4. data from regionprops\_table of one of the field of views

**Analysis of results**

**Filtering the data**

Looking at the area of individual labelled regions, one can see that some regions have very small areas and some regions have very big areas. The small areas are background noise while the big areas are more than 1 cell being clumped together, both of which are data that we do not want to include in our analysis. Therefore, to filter these outliers, we find the mean of the area and only include the areas that are within 2 standard deviations of the mean.

**plotting**

To visualise the distribution of the fluorescent signal of mVenus and mCherry of the samples and its respective area, we plotted a scatter plot of fluorescent intensity against area for both 0μM and 100μM vanillic acid in the test strain. This allows us to visualise the relationship between area and fluorescent signal and visualise the area and fluorescent intensity of individual cells. As we can see from the scatter plot that the fluorescent signal and the area is independent of each other.

![Scatter plot for 0μM vanillic acid.](https://static.igem.wiki/teams/4491/wiki/engineering/mcherry-mvenus-0um-scatter-new.png)

Figure 5. Scatter plot for mVenus and mCherry intensity of 0μM vanillic acid in test strain

![Scatter plot for 100μM vanillic acid.](https://static.igem.wiki/teams/4491/wiki/engineering/mcherry-mvenus-100um-scatter-new.png)

Figure 6. Scatter plot for mVenus and mCherry intensity of 100μM vanillic acid in test strain

To visualise the distribution of fluorescent signal of mVenus and mCherry based on counts, we have decided to plot histograms for the counts of fluorescent intensities. We have plotted 2 types of histograms - mCherry and mVenus on the same graph for the same cell type and mVenus of different cell type plotted on the same graph. The first allows us to see the difference between mCherry and mVenus expression while the second allows us to visually compare the effect of Vanillic acid on the system.

![Histogram plot of the mCherry and mVenus intensity of test strain with 0μM Vanillic acid](https://static.igem.wiki/teams/4491/wiki/engineering/mcherry-mvenus-0um-his-new.png)

Figure 7. Histogram plot of mCherry and mVenus intensity of the test strain with 0μM vanillic acid. The mean of the the intensity of mCherry is 4153 with a standard deviation of 1026. The mean of the intensity of mVenus is 4769 with a standard deviation of 1272.

![Histogram plot of the mCherry and mVenus intensity of test strain with 100μM Vanillic acid](https://static.igem.wiki/teams/4491/wiki/engineering/mcherry-mvenus-100um-his-new.png)

Figure 8. Histogram plot of mCherry and mVenus intensity of the test strain with 100μM vanillic acid. The mean of the the intensity of mCherry is 3279 with a standard deviation of 486. The mean of the intensity of mVenus is 7259 with a standard deviation of 1471.

![Histogram plot of the test strain of mVenus intensity with 0μM and 100μM Vanillic acid](https://static.igem.wiki/teams/4491/wiki/engineering/0um100um-mvenus.png)

Figure 9. Histogram plot of mVenus intensity of the test strain with 0μM and 100μM vanillic acid. The mean of the mVenus intensity of 0μM vanillic acid is 4769 with a standard deviation of 1272 while the mean of the mVenus intensity of 100 μM vanillic acid is 7259 with a standard deviation of 1471.

The above analysis were all performed at a single timepoint during the experiment. To further characterise MegaT, we did the same analysis on an overnight time-lapse imaging to see where the fluorescent intensity of the cells changes with time. As the cells dried up overnight and died, we splice the time period of the experiment within the period where the cells are not dried up and plotted a time series data of how the mean of the fluorescent intensity change with time. As we can see from the graph, the mean intensity of the cells with nFc in 100μM of vanillic acid is always higher than that of the cells with nFc in 0 μM of vanillic acid.

![Time series data of MegaT characterisation](https://static.igem.wiki/teams/4491/wiki/engineering/timeseries-megat.png)

Figure 10. Time series data of MegaT characterisation showing the mean of fluorescent intensity with time

**Statistical Testing**

As we are interested in comparing the mean intensities of mVenus expression between 0µM and 100µM Vanillic acid, we perform a *t-test* between the 2 samples and confirm that the 2 means are statistically significantly different from each other.

**Conclusion**

The result shows that MegaT can respond to the relative changes of VanR, it shows us that it is a desirable candidate to report the relative amount of the species it is in the same transcription unit with. As we are trying out a combinatorial approach to test for the antithetic integral controller with the best performance, we have decided to design MegaT with a different ribosome binding sites, matching that of the ribosome binding site of species X in our project.

# Design 2

Due to what we have learnt above, we have designed 2 other MegaT, one with RBS B0033 (weak) and the other with RBS B0034 (strong). The following figure shows the design of all the MegaT, showing that this can be put into a package to allow users of the antithetic circuit to choose the MegaT based on the RBS of species X.

![Design of a family of MegaT](https://static.igem.wiki/teams/4491/wiki/engineering/megat-family.png)

Figure 11. Design of a family of MegaT to allow users to choose which one to use for their circuit

# Autoregulatory Negative feedback

Negative feedback is a type of circuit design motif that exists in nature to improve system response and reject disturbances. To know more about how a negative feedback motif acts to reject disturbances, please refer to the [modelling](https://2022.igem.wiki/cambridge/model#types) page. The purpose of the experiment is to characterise the autonegative feedback circuit by seeing how it adapts to constant disturbance as a perturbation. Using the mother machine, a microfluidic device, we are able to control the medium the cells are cultured in. We image the cells with time-lapse microscopy at 1 µM Vanilic acid for 3 hours, making sure that the steady state value is reached. We then perturb the system by switching the culture medium to 10 µM vanillic acid for another 3 hours to see how the system adapts.

## Design (cycle 1)

We have designed an autonegative feedback loop (nFc) to study the effect of adaptation. An autoregulatory negative feedback refers to a motif that involves a species feeding back to downregulate itself. The feedback species we have chosen is VanR and the promoter we have chosen is P\_VanCC, the effect of VanR on P\_VanCC at different concentration of Vanillic acid pf inducer is well characterised by Meyer *et al.,* (2019). Essentially, VanR will act on P\_VanCC, repressing its production, following a Hill Function dynamics. In order to characterise the amount of vanR, we have created an operon (MegaT) with a RBS, mVenus and Terminator after *VanR* coding sequence (CDS). The detailed design on MegaT is discussed [above](https://2022.igem.wiki/cambridge/engineering#MegaT).

![Design of the negative feedback circuit](https://static.igem.wiki/teams/4491/wiki/engineering/negative-feedback-32-new1.png)

Figure 12. Circuit diagram of the autoregulatory negative feedback system with RBS B0032. X: feedback species (in this case VanR), R: Reporter (in this case mVenus)

## Build (cycle 1)

We perform JUMP assembly to clone the transcription unit into pJUMP27-1A using the enzyme BsaI to clone the transcription unit into the main cloning site. For the protocol we used for JUMP assembly, please refer to the [Experiments](https://2022.igem.wiki/cambridge/experiments#jump) page. For details of how JUMP Golden Gate Assembly works, please refer to the [Rationale](https://2022.igem.wiki/cambridge/design#jump) page.

After Golden Gate Assembly, the construct is then chemically transformed into DH5a cloning strain and plated out. For details of the protocol, please refer to the [Chemical transformation](https://2022.igem.wiki/cambridge/experiments#chem_trans) protocol on the Experiments page.

After overnight incubation on Kanamycin selection plates, we want to confirm the construct we want exists in the colony. To do this, we perform colony PCR and gel electrophoresis on a non-fluorescent colony that does not contain superfolder GFP (sfGFP) and check whether it has the correct length of amplicon after colony PCR (cPCR). The reason we are choosing non-fluorescent colonies is because sfGFP is the drop out cassette of the JUMP vector. Therefore, if the transcription unit is successfully inserted into the JUMP vector, the sfGFP will be lost, thus the colony will not be fluorescent. For the details of the protocol, please refer to the [colony PCR](https://2022.igem.wiki/cambridge/experiments#cPCR) and the [gel electrophoresis](https://2022.igem.wiki/cambridge/experiments#gel) protocol on the Experiments page.

For the colony with the correct cPCR amplicon length on gel electrophoresis, the colony grown overnight and glycerol stocks and Miniprep are done the next day. For details of the protocol, please refer to the [glycerol stocks](https://2022.igem.wiki/cambridge/experiments#glycerolstocks) and [Miniprep protocol](https://2022.igem.wiki/cambridge/experiments#miniprep) on the Experiments page.

For the construction of the autoregulatory negative feedback circuit, colony PCR is a good indication that cloning is successful. The band length of the autoregulatory negative feedback circuit is about 2000bp while for superfolder GFP is about 1300bp. Moreover, to make sure that there is no mutation within the construct, the sequence has to be verified through sequencing. Sequence verification in this case is very important because MegaT contains a fluorescent protein, mVenus, which may fail to report on the amount of VanR if it contains mutations.

## Test (cycle 1)

To test the effect of the negative feedback circuit in response to perturbation, we have decided to conduct the experiment in a mother machine.

**Fabrication of the Microfluidic Device**

A mother machine is a microfluidic device that is made up of a feeding lane and trenches on its side. The design of the mother machine allows cells to be captured within the trenches. The first cell that gets captured in the trench is the "Mother" cell. The feeding lane allows fresh medium to flow through it constantly, providing nutrients to the cells inside the trenches of the mother machine. The following figure shows a mother machine under the microscope where we can see the trenches on the side of the feeding lane.

![Mother Machine under microscope](https://static.igem.wiki/teams/4491/wiki/engineering/2210106-20x-1m-igem22-str-mm-checkuppng.png)

Figure 13. The design of Mother Machine under a microscope image

**Preparation of Overnight Culture**

The following table shows the strain of cells and culture conditions overnight we have used for the characterisation of the negative feedback circuit. The test strain we have used is called SB7, a strain with mCherry integrated into chromosomal DNA. We have used *E. coli* SB6, a strain with mVenus integrated into the chromosomal DNA as a positive control and *E. coli* SB5, a strain with CFP integrated into the chromosomal DNA as a positive control.

| Strain | Plasmid | Medium (Overnight culture) | Antibiotics | Volume | Purpose |
| --- | --- | --- | --- | --- | --- |
| *E. coli* K-12 MG1655 7740 (ΔmotA, glmS::PRNA1-mCherry-mKate2) [aka. SB7] | nFc | EZRDM - 0.1% pluronic | Kanamycin | 2mL | Test strain |
| *E. coli* (SB7) | nFc | EZRDM - 0.1% pluronic - 100µM Vanillic Acid | Kanamycin | 2mL | Pre-test sample to check full expression fluorescence level before experiment start under the microscope to test required microscope settings |
| *E. coli* (E. coli MG1655 7740 ΔmotA attTn7::PrpsL-SCFP3A) [aka. SB5] | No plasmid | EZRDM - 0.1% pluronic | Kanamycin | 2mL | Negative control |
| *E. coli* K-12 MG1655 7740 (ΔmotA, attTn7::PrpsL-Venus) [aka. SB6] | No plasmid | EZRDM - 0.1% pluronic | Kanamycin | 2mL | Positive control |
Table 3. Overnight culture for characterisation of negative feedback circuit (nFc)

**Microscope settings**

As we need to visualise the cells and the mother machine under the microscope (Nikon TI2 Eclipse), we have to have appropriate microscope settings to image the cells.

| Channel | Reporter | Wavelength | Purpose |
| --- | --- | --- | --- |
| phase contrast | NA | 705nm | show the presence of cells |
| mCherry | mCherry | 610nm | Show the presence of SB7 - all SB7 cells have mCherry integrated into their chromosome  For cell segmentation |
| YFP | mVenus | 530nm | To show the concentration of the reporter  To show positive control  For cell segmentation |
| CFP | CFP | 475nm | Show negative control  For cell segmentation |
Table 4. Microscope settings for the characterisation of negative feedback circuit (nFc)

Note: Segmenting cells with phase contrast alone can be difficult due to low contrast. Therefore, we require a fluorescent signal image for cell segmentation.

Microscope images were taken every 3 minutes to allow for good time resolution at 9 different field of views along the trenches of the mother machine.

Below is a video showing the microscope in action, imaging the cells in different channels!

**Testing in the Mother Machine**

We have prepared 2 media which was put into the mother machine in the first 3 hours and last 3 hours of the experiment respectively. The first media we have made consists of EZRDM, pluronic and 1μM Vanillic acid. The second media we have made consists of EZRDM, pluronic and 10μM Vanillic acid. The explanation of the choice of the media is explained below.

![cells in mother machine (early in the experiment)](https://static.igem.wiki/teams/4491/wiki/engineering/220927-igem22-fov9-t017.png)

Figure 14. Microscope image of cells inside a mother machine in the beginning of the experiment

*Explanation*

With 1μM vanillic acid added, vanR produced represses on p\_vanCC, inhibiting mVenus production. After the circuit reaches its steady state, we introduce the perturbation as 10μM vanillic acid. As this is a negative feedback loop but not robust perfect adaptation, 10μM of vanillic acid should act to inhibit the repression of vanR on p\_vanCC so the signal of mVenus is expected to increase. The concentration of vanillic acid chosen for the experiment is based on the characterisation from Meyer *et al.,* (2019). To allow the system to have a large dynamic range, we have chosen concentrations of vanillic acid that lie on the lower end and the higher end of the slope of the Hill curve. As we are interested in a positive perturbation, we started the system off with a vanillic acid concentration of 1μM then use 10μM vanillic acid as a perturbation.

## Learn (cycle 1)

Through looking at the data from the microscope as a quick analysis, we have realised that only the cells in the middle of the field of view survived but those on the side are all dead at the end of the experiment (Figure 4). Moreover, this happens consistently in all the field of views we have captured. We proceed on looking at the time-lapse images and realise that the cells on the periphery of the field of views are slowly dying out as the experiment proceeds. As the experiment requires using high energy photons to detect CFP emission from SB6 strain, we suspect that this effect we saw from such quick-analysis is due to the effect of phototoxicity. As the different field of views are close together and the area that the excitation light shines on is wider than the field of view itself, the cells on the periphery are excited for more than once during every cycle of taking images, leading to their death.

![cells in mother machine (late in the experiment)](https://static.igem.wiki/teams/4491/wiki/engineering/220927-igem22-fov9-t114.png)

Figure 15. Microscope image of cells inside a mother machine in the middle of the experiment, showing that cells in the periphery of the field of view has lost its fluorescence and died

As the cells in the middle of the field of view are largely unaffected, we looked at the time-lapse images to see whether we see the effect we expect after the addition of vanillic acid. As we are going through the images, we see that the cells increases in intensity of mVenus as expected. However, towards the end of the experiment, we see signs that the cells are “struggling” and getting dimmer, probably also due to the phototoxicity brought about by the UV light that excites CFP.

To show that the fluorescent intensities of cells in the presence of perturbation of vanillic acid, we plotted a time series data of the cells that survived the imaging. However, as it is evident that some cells are dying as the imaging goes on and lost their fluorescent, we have limited the time range we analyse the time series data. The following figure shows the change in intensity of 3 cells as an example to see how the intensity of the cells have increased with the change of medium from 1µM of vanillic acid to 10µM of vanillic acid.

![time series data](https://static.igem.wiki/teams/4491/wiki/engineering/mm-3cells-new.png)

Figure 16. Time series data of 3 cells of the mother machine experiment, showing an increase in fluorescent intensity in response to the addition of vanillic acid.

To visualise the effect of vanillic acid on the fluorescent intensity, the distribution of fold change of fluorescent intensites of all the cells is plotted below. The fold change is calculated by the ratio of the intensity of the end point and the intensity of the starting point. Apart from the fold change, the halfway point, the time point where half of the maximum intensity is reached, is also being plotted out as a distribution to look at the response time of the circuit.

![fold change](https://static.igem.wiki/teams/4491/wiki/engineering/fold-change-new.png)

Figure 17. The distribution of fold change in the experiment. The mean fold change is 1.35 with a standard deviation of 0.3.

![response time](https://static.igem.wiki/teams/4491/wiki/engineering/response-time-new1.png)

Figure 18. The distribution of response time in the experiment. The mean of the response time is 15.1 time points with a standard deviation of 5.3 time points.

## Design (cycle 2)

Learning from the data obtained from the mother machine experiment, we have decided to iterate through another cycle by doing simulations of the autoregulatory negative feedback system to further improve system response. Through doing the simulation, we find that increasing the strength of the production rate of VanR reduces the system deviation from the setpoint in response to the same perturbation. Therefore, we have designed the next negative feedback circuit, replacing the RBS with B0034, a stronger RBS than B0032 to investigate whether it can improve system dynamics. Below is the figure showing the changes we are trying to implement for this new cycle.

![new negative feedback design](https://static.igem.wiki/teams/4491/wiki/engineering/negative-feedback-34-new1.png)

Figure 19. Circuit diagram of the autoregulatory negative feedback system with RBS B0034 to increase RBS strength

![Simulation of different RBS strengths](https://static.igem.wiki/teams/4491/wiki/engineering/compare-negfeedback.png)

Figure 20. Simulation showing a stronger production of vanR improves system response

We have also noted that the data we have obtained from the first cycle may not be reliable as the phototoxicity damaged the cells. Therefore, for the next iteration, we plan to perform the experiment by manipulating the fieldstop, ensuring that the excitation light only illuminate the field of view itself but not the area out of the field of view, avoid illuminating cells in the periphery of the field of view more than once in each cycle.

# Downstream site insertion

In the main project, we want to create a perturbation circuit to test our whether the antithetic integral controller can robustly adapt. As building the antithetic integral controller together with the perturbation circuit requires 5 transcription units and a level 2 plasmid can accommodate 4 transcription units at the same time, we have decided to insert the remaining transcription unit of the perturbation circuit into the downstream site of pJUMP47-2A.

## Design (Cycle 1)

At the first instance, we have decided to look at the protocol given in the original paper of JUMP Golden Gate Assembly (Valenzuela-Ortega *et al*., 2021) where the authors make use of a 2-step assembly method to perform a downstream site integration. In the first step, basic parts required for the linear transcription unit for insertion is assembled and at the time the destination vector is digested separately with BbsI and dephosphorylated. After this, in the second step, the ligated mixture and the digested and dephosphorylated backbone is put together in the same reaction mixture and is ligated.

As we believe the protocol used in the paper was a bit too complicated for us, we have decided to design our own protocol to insert the linear assembly into the downstream site. We have designed a linear Golden Gate Assembly pipeline to create an insert that has adapter on both the 5’ and the 3’ end with the recognition site of BbsI.

The 5’ adapter is divided into 3 parts - the primer amplification region, the BbsI recognition site with the correct overhangs and the BsaI recognition site with the correct overhangs. The primer amplification region allows the same primer design to be able to be compatible with the amplification of all linear Golden Gate assembly products. The BbsI recognition site allows the Golden Gate assembled product to be inserted into the downstream site. The BsaI recognition site allows linear Golde Gate assembly to occur, forming a linear ligated product of 5’ adapter, promoter, RBS, CDS, Terminator and 3’ adapter.

The following figure shows the design of the adapters

![5' adapter](https://static.igem.wiki/teams/4491/wiki/engineering/5-adapter.png)

Figure 21. Design of the 5' adapter

![3' adapter](https://static.igem.wiki/teams/4491/wiki/engineering/3-adapter.png)

Figure 22. Design of the 3' adapter

## Build (Cycle 1) As the adapter sequence is less than 50bp, we have ordered each adapter sequence as 2 strands of oligos. The adapters are first annealed together into double-stranded DNA with the oligo annealing protocol. Please refer to the Experiments page for the protocol of [oligo annealing](https://2022.igem.wiki/cambridge/experiments#OA). After obtaining the adapter in the double-stranded DNA form, the adapter is molaritised into 25nM. By diluting the necessary parts of the linear transcription unit (J23106, B0032, TetR, ECK120029600) into 15nM, we then perform linear Golden Gate Assembly using the [linear Golden Gate Assembly pipeline](https://2022.igem.wiki/cambridge/experiments#linearGGA) specified in the Experiments page. After linear Golden Gate assembly, the product is then amplified with Q5 PCR then validated through gel electrophoresis whether it shows a correct length on the gel. If it does, we purify the linear Golden Gate product. If not, we have to repeat the above steps. After purification, the linear transcription unit is ready to be integrated into the downstream site. For this, we make use of the [downstream site integration](https://2022.igem.wiki/cambridge/experiments#jump) protocol in the experiments page. Subsequent steps of JUMP assembly is the same for downstream site integration compared to cloning into the main site. For details, please refer to the [cloning workflow](https://2022.igem.wiki/cambridge/experiments#workflow) described in the Experiments page. Test (Cycle 1) To make sure that the downstream site is inserted, we have to formulate ways to test and confirm that the integration is successful. Normally, there are 3 ways to validate integration into the cloning site - visual inspection through fluorescence, colony PCR and sequencing. While we can perform visual inspection on inserts into the main cloning site due to the presence of a superfolder GFP dropout cassette, the downstream site integration does not displace any dropout cassette so when looking at the colonies, we cannot use visual inspection to decide which colonies will have the downstream site integrated thus which ones to pick. As a result, the second way to confirm this is through colony PCR. To increase the chance of success, we have decided to pick about 12 colonies, a lot more than the normal cloning workflow into the mainstream site. Figure x shows an example of the gel with 3 lanes being the downstream site integrated into the Level 2 acceptor. As we can see, the band length shown in the gel is about 2000, an expected band length produced through colony PCR. After confirming the band length on the gel, we confirm the construct through sequence verification with Sanger sequencing. ![DSS gel](https://static.igem.wiki/teams/4491/wiki/engineering/dss-gel-new.png) Figure 23. Gel electrophoresis showing the correct size bands for downstream site integration into Level 2 acceptor (pJUMP47-2A) Learn (Cycle 1) After submitting the plasmid for sequencing, we have confirmed that the downstream site has been inserted successfully and have proved that inserting a linear transcription unit into the downstream site through the design of adapters is possible. The following figure shows the sequence alignment of the sequencing results and the design of our linear Golden Gate Assembly into the downstream site. ![DSS sequence verification](https://static.igem.wiki/teams/4491/wiki/engineering/dss.png) Figure 24. Sequence verification of downstream site integration We have also submitted an entry of the Downstream site perturbation circuit onto the Registry! Registry No. [BBa\_K4491145](http://parts.igem.org/Part%3ABBa_K4491145) Design (Cycle 2) Through doing the downstream site cloning, we have also realised how inconvenient it is to be unable to select for colonies through visual inspection because we have to pick a larger number of colonies for colony PCR. As a result, we think of replacing the current terminator (ECK120029600) with MegaT\_RFP, a variant of MegaT that we have decided to allow for modularity of our system (see [Implementation](https://2022.igem.wiki/cambridge/implementation) page for more details). As MegaT\_RFP contains a whole transcription unit and appear red by the naked eye, it can act as a selection marker that reduces the number of colonies we need to pick for colony PCR. The following figure shows the selection plate with colonies containing MegaT\_RFP on it, as we can see, the colonies are visibly red, allowing red-green selection. ![MegaT_RFP plate](https://static.igem.wiki/teams/4491/wiki/engineering/megat-rfp-plate-rotate.jpg) Figure 25. Selection plates with colonies containing MegaT\_RFP in it, showing that it is visibly red References 1. Meyer, A.J., Segall-Shapiro, T.H., Glassey, E. *et al*. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nat Chem Biol 15, 196–204 (2019). https://doi.org/10.1038/s41589-018-0168-3 2. Chen, YJ., Liu, P., Nielsen, A. *et al.* Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods 10, 659–664 (2013). https://doi.org/10.1038/nmeth.2515 3. Marcos Valenzuela-Ortega, Christopher French, Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology, Synthetic Biology, Volume 6, Issue 1, 2021, ysab003, https://doi.org/10.1093/synbio/ysab003

[Cloning Workflow](#workflow)

[Chemical Transformation](#chem_trans)

[Colony PCR](#cPCR)

[Gel electrophoresis](#gel)

[Electroporation](#electroporation)

 JUMP Golden Gate Assembly ▼

[Level 0](#lvl0)

 [Level 1](#lvl1)

 [Level 2](#lvl2)

 [DSS](#dss)

[Linear Golden Gate Assembly](#linearGGA)

[Making glycerol stocks](#glycerolstocks)

[Miniprep](#miniprep)

[Oligo Annealing](#OA)

[Q5 PCR](#q5)

[PCR purification](#purification)

[Making LB agar selection plates](#agar)

[Making LB broth medium](#broth)

Protocols

---

## Cloning Workflow

Cloning is a major part of our main project to create the antithetic integral controller. The figures below show the workflow of our project. For detailed protocol, please refer to protocols stated below.

![Level 0 cloning workflow.](https://static.igem.wiki/teams/4491/wiki/experiments/level0workflow-small.png)

Figure 1. Workflow of Level 0 cloning from ordered DNA parts

![Lvel 1/2 cloning workflow.](https://static.igem.wiki/teams/4491/wiki/experiments/level12workflow-small.png)

Figure 2. Workflow of Level 1 and Level 2 cloning

## Experimental Protocols

### Chemical Transformation

Purpose: to transform DNA (plasmid) into competent cells

Material
LB broth, ice, selection plates, SOC, spreaders

Preperation steps

1. Obtain ice

2. Turn the heat block on

3. Put SOC in room temperature

4. Place selection plates into 37°C incubation

Methods

1. Thaw 50µL competent *E. coli* cells on ice for 10 minutes

2. Split the 50muL of bacteria into 3 tubes of 16uL or 4 tubes of 12uL and transfer to the labelled tubes placed on ice beforehand.

3. Add 2.5muL of Golden Gate Assembly product into split competent cells

4. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex

5. Place the mixture on ice for 30 minutes. Do not mix

6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix

7. Place on ice for 5 minutes. Do not mix

8. Pipette 950 µl of room temperature SOC into the mixture

9. Incubate at 37°C and 200-250 rpm for 60 minutes

10. Mix the cells thoroughly by flicking the tube and inverting

11. Spread the plates

* 50µL of transformation mixture onto one plate

* Remaining transformation mixture onto another plate

* Spread with spreader

12. Incubate overnight at 37°C with plates upside down or incubate at 27°C for 48 hours

### Colony PCR

Purpose: To amplify the desired construct inside a colony ready for gel electrophoresis

Material
Dream Taq Green Master Mix, forward primer (10µM), reverse primer (10µM), selectin plates, water, thermocycler, PCR tubes/plates

| cPCR reaction contents | 1X |
| --- | --- |
| DreamTaq Green Master Mix | 10 µL |
| Forward primer (10 µM) | 1 µL |
| Reverse primer (10µM) | 1 µL |
| Resuspended colony | 1 µL |
| water (nuclease free) | 7 µL |
| Total volume | 20 µL |
Table 1. Material required for making the master mix for colony PCR

Methods

1. Add a single colony of cells to 13 µL of PBS./li>
   - Put 1muL of the resuspended colony into the above recipe of PCR mixture

   - Spin down the PCR mixture with a centrifuge

   - Put the remaining 12uL PBS with resuspended colony in the fridge

   - Incubate in the thermocycler

   * Initial denaturation: 95°C 1-3 minutes

   * Cycles (35 cycles)
   + Denaturation: 95°C 30 seconds
   + Annealing: Tm 30 seconds (primer dependent, calculated with NEB Tm calculator)
   + Extension: 72 °C (length dependent: 30s/kb)* Final extension: 72°C 5-15 minutes
   * Hold: 4°C Indefinitely- Gel electrophoresis

   - Transfer the 12uL of PBS with resuspended colony into LB liquid culture for overnight growth given colony PCR yields positive results after gel electrophoresis

### Gel electrophoresis

Purpose: To investigate whether the DNA sample is of the correct length

Material

1X TAE, Agarose, SYBR Safe, Loading Buffer, Hyperladder, Casting tray, well combs, Voltage source, gel tank, suitable light source, microwave

Methods
**Making agarose gel**

1. For 1% gel, measure 1g of agarose per 100mL TAE and mix the appropriate amount of agarose and TAE/li>
   - Microwave and mix for 1-3 minutes until the agarose is completely dissolved

   * Do not overboil

   * Microwave in pulses and swirl occasionally- Cool for about 5 minutes

   - Add 1µL/10mL TAE of SYBR Safe and swirl until well mixed

   - Pour slowly the agarose into a gel tray with the well comb in place

   - Use a pipette tip to remove any bubbles or move any bubbles to the corner

   - Wait for about 30 minutes for the gel to set at the room temperature

**Loading gel and electrophoresis**

1. Add 1µL of loading buffer to 5 µL of DNA sample

* DreamTaq Green Master Mix contains loading dye thus this step is not required

2. Place the set gel into the gel tank with the comb side at the negative end of the gel tank and remove the comb carefully

3. Fill the gel tank with TAE to cover the comb holes

4. Add 8µL of 1kB hyperladder to the first well

5. Carefully load the remaining samples

6. Set the voltage to about 90V and press run

7. Wait until the dye is approximately 75-80% way down

8. Turn off and remove electrodes

9. Remove gel and visualise under blue light source

### Electroporation

Purpose: To make incompetent cells electrically competent

Material

Electroporator (BioRad GenePulser), SOC, ice, e-cuvette, selection plates, spreaders

Methods
**Glycerol washing**

1. Inoculate a single colony of incompetent cells into 2 mL LB containing the appropriate antibiotics and incubate at 37°C overnight

2. Dilute overnight culture 1 mL into 25 mL LB containing appropriate antibiotics and shake at 37 °C until OD600 = 0.6-0.8 (1.5 hours to 3 hours)

3. Transfer the culture into 50 mL falcon tube

4. Spin down cells at 5000 rpm at 4°C for 10 minutes and resuspend pallet in 25 mL ice-cold 10% glycerol

5. Repeat step 4

6. Spin down cells at 5000 rpm at 4°C for 10 minutes

7. Resuspend pallet in 1 mL ice-cold 10% glycerol

8. Spin down cells at 14000 rpm at 4°C for 1 minute

9. Resuspend pallet in 250µL ice-cold 10% glycerol

10. Aliquot into ice-cold cyro tubes (Each 50µL) and store at -80°C

**Electroporation**

1. Thaw the prewashed glycerol stocks (50uL) on ice

2. Chill the e-cuvettes and slider on ice

3. Put 1µL of DNA into the stock tube containing glycerol stocks and wait for 5 minutes on ice

4. Transfer the entire volume inside the stock tube into a e-cuvette, make sure that the liquid is in contact with the metal wall

5. Wipe the e-cuvette and the slider to make sure no water droplet is on the wall

* If there is still water droplet, the pulse will cause spark which will damage the cells

6. Put the e-cuvette into the slider and slide the slider with the e-cuvette into the chamber of the electroporator

7. Start electroporation by pressing on the pulse button and hold for 5 seconds until a buzzing tone is heard. Release and slide the slider out of the chamber

8. Add 500µL of SOC into the e-cuvette immediately

9. Carefully transfer all the mixture from the e-cuvette into a 1.5mL tube

10. Incubate at 37°C and 200-250 rpm for 60 minutes

11. Spread the plates

* 50µL of transformation mixture onto one plate

* Remaining transformation mixture onto another plate

* Spread with spreader

12. Incubate overnight at 37°C with plates upside down or incubate at 27°C for 48 hours

### JUMP Golden Gate Assembly

Purpose: To perform cloning to put DNA pieces together at a desired sequence

#### Level 0

Material

T4 ligase buffer, T4 ligase enzyme, BsmBI-v2/Esp3I, water, DNA inserts, DNA destination vector, PCR tubes

If using BsmBI-v2

| JUMP Level 0 Contents | 1X (10µL) |
| --- | --- |
| T4 ligase buffer | 1 µL |
| T4 ligase enzyme | 0.5 µL |
| BsmBI-v2 | 0.5 µL |
| water (Nuclease free) | 6 µL |
| Desination vector (7.5nM) | 1 µL |
| Insert (15nM) | 1 µL |
| Total volume | 10 µL |
Table 2. Material required for Level 0 JUMP Golden Gate Assembly with BsmBI-v2

**Thermocycler conditions**
*Non-overnight assembly protocol*

* 42°C 15 minutes

* 30 cycles

+ 42°C 3 minutes

+ 16°C 3 minutes

* 55°C 15 minutes

* 80°C 5 minutes

* 4°C hold

*Overnight assembly protocol*

* 42°C 15 minutes

* 60 cycles

+ 42°C 5 minutes

+ 16°C 5 minutes

* 55°C 60 minutes

* 80°C 5 minutes

* 4°C hold

If using Esp3I

| JUMP Level 0 Contents | 1X (10µL) |
| --- | --- |
| T4 ligase buffer | 1 µL |
| T4 ligase enzyme | 0.5 µL |
| Esp3I | 1 µL |
| water (Nuclease free) | 5.5 µL |
| Desination vector (7.5nM) | 1 µL |
| Insert (15nM) | 1 µL |
| Total volume | 10 µL |
Table 3. Material required for Level 0 JUMP Golden Gate Assembly with Esp3I

**Thermocycler conditions**
*Non-overnight assembly protocol*

* 37°C 3 minutes

* 20 cycles

+ 37°C 2 minutes 30 seconds

+ 16°C 3 minutes

* 50°C 5 minutes

* 80°C 10 minutes

* 4°C hold

*Overnight assembly protocol*

* 37°C 3 minutes

* 40 cycles

+ 37°C 9 minutes

+ 16°C 12 minutes

* 55°C 5 minutes

* 80°C 10 minutes

* 4°C hold

#### Level 1

Material

T4 ligase buffer, T4 ligase enzyme, BsaI-HF, water, DNA inserts, DNA destination vector, PCR tubes

| JUMP Level 1 Contents | 1X (10µL) |
| --- | --- |
| T4 ligase buffer | 1 µL |
| T4 ligase enzyme | 0.5 µL |
| BsaI-HF | 0.5 µL |
| water (Nuclease free) | 3 µL |
| Desination vector (7.5nM) | 1 µL |
| Insert 1 (15nM) | 1 µL |
| Insert 2 (15nM) | 1 µL |
| Insert 3 (15nM) | 1 µL |
| Insert 4 (15nM) | 1 µL |
| Total volume | 10 µL |
Table 4. Material required for Level 1 JUMP Golden Gate Assembly with BsaI-HF

**Thermocycler conditions**
*Non-overnight assembly protocol*

* 37°C 3 minutes

* 20 cycles

+ 37°C 2 minutes 30 seconds

+ 16°C 3 minutes

* 50°C 5 minutes

* 80°C 10 minutes

* 4°C hold

*Overnight assembly protocol*

* 37°C 3 minutes

* 40 cycles

+ 37°C 9 minutes

+ 16°C 12 minutes

* 55°C 5 minutes

* 80°C 10 minutes

* 4°C hold

#### Level 2

Material

T4 ligase buffer, T4 ligase enzyme, BsmBI-v2/Esp3I, water, DNA inserts, DNA destination vector, PCR tubes

If using BsmBI-v2

| JUMP Level 2 Contents | 1X (10µL) |
| --- | --- |
| T4 ligase buffer | 1 µL |
| T4 ligase enzyme | 0.5 µL |
| BsmBI-v2 | 0.5 µL |
| water (Nuclease free) | 3 µL |
| Desination vector (7.5nM) | 1 µL |
| Insert 1 (15nM) | 1 µL |
| Insert 2 (15nM) | 1 µL |
| Insert 3 (15nM) | 1 µL |
| Insert 4 (15nM) | 1 µL |
| Total volume | 10 µL |
Table 5. Material required for Level 2 JUMP Golden Gate Assembly with BsmBI-v2

**Thermocycler conditions**
*Non-overnight assembly protocol*

* 42°C 15 minutes

* 30 cycles

+ 42°C 3 minutes

+ 16°C 3 minutes

* 55°C 15 minutes

* 80°C 5 minutes

* 4°C hold

*Overnight assembly protocol*

* 42°C 15 minutes

* 60 cycles

+ 42°C 5 minutes

+ 16°C 5 minutes

* 55°C 60 minutes

* 80°C 5 minutes

* 4°C hold

If using Esp3I

| JUMP Level 2 Contents | 1X (10µL) |
| --- | --- |
| T4 ligase buffer | 1 µL |
| T4 ligase enzyme | 0.5 µL |
| Esp3I | 1 µL |
| water (Nuclease free) | 2.5 µL |
| Desination vector (7.5nM) | 1 µL |
| Insert 1 (15nM) | 1 µL |
| Insert 2 (15nM) | 1 µL |
| Insert 3 (15nM) | 1 µL |
| Insert 4 (15nM) | 1 µL |
| Total volume | 10 µL |
Table 6. Material required for Level 2 JUMP Golden Gate Assembly with Esp3I

**Thermocycler conditions**
*Non-overnight assembly protocol*

* 37°C 3 minutes

* 20 cycles

+ 37°C 2 minutes 30 seconds

+ 16°C 3 minutes

* 50°C 5 minutes

* 80°C 10 minutes

* 4°C hold

*Overnight assembly protocol*

* 37°C 3 minutes

* 40 cycles

+ 37°C 9 minutes

+ 16°C 12 minutes

* 55°C 5 minutes

* 80°C 10 minutes

* 4°C hold

#### Downstream site (DSS) integration

Material

T4 ligase buffer, T4 ligase enzyme, BbsI, water, linear ligated DNA, DNA destination vector, PCR tubes

| JUMP DSS integration Contents | 1X (10µL) |
| --- | --- |
| T4 ligase buffer | 1 µL |
| T4 ligase enzyme | 0.5 µL |
| BbsI | 1 µL |
| water (Nuclease free) | 5.5 µL |
| Desination vector (7.5nM) | 1 µL |
| linear ligated DNA (15nM) | 1 µL |
Table 4. Material required for DSS JUMP Golden Gate Assembly with BbsI

**Thermocycler conditions**
*Non-overnight assembly protocol*

* 37°C 3 minutes

* 20 cycles

+ 37°C 2 minutes 30 seconds

+ 16°C 3 minutes

* 50°C 5 minutes

* 80°C 10 minutes

* 4°C hold

*Overnight assembly protocol*

* 37°C 3 minutes

* 40 cycles

+ 37°C 9 minutes

+ 16°C 12 minutes

* 55°C 5 minutes

* 80°C 10 minutes

* 4°C hold

### Linear Golden Gate Assembly

Purpose: to linear ligate fragments of DNA together to form transcription units and adapters on either sides for subsequent assemblies

**Linear ligation**
Material
DNA adapter, DNA fragments, DNA ligase buffer, DNA ligase, BsaI-HF, nuclease free water, PCR tubes, thermocycler

| linear Golden Gate Assembly Contents | 1X (10µL) |
| --- | --- |
| T4 ligase buffer | 1 µL |
| T4 ligase enzyme | 0.5 µL |
| BsaI-HF | 0.5 µL |
| water (Nuclease free) | 2 µL |
| 5' adapter (25nM) | 1 µL |
| Promoter (15nM) | 1 µL |
| RBS (15nM) | 1 µL |
| CDS (15nM) | 1 µL |
| Terminator (15nM) | 1 µL |
| 3' adapter (25nM) | 1 µL |
| Total volume | 10 µL |
Table 7. Material required for linear Golden Gate Assembly with BsaI-HF

Methods
*Thermocycler Conditions*

* Cycles (9 cycles)

+ 37°C 3 minutes

+ 16°C 4 minutes

* 50°C 5 minutes

* 4°C hold

**Post linear ligation amplication**
Materials
Q5 PCR Hot Start Master Mix, forward primer, reverse primer, nuclease free water, Golden Gate Assembly product, PCR tube, thermocycler, materials for [gel electrophoresis](https://2022.igem.wiki/cambridge/experiments#gel)

| Q5 PCR amplication contents | 1X (50µL) |
| --- | --- |
| 2X Q5 Hot Start Master Mix | 25 µL |
| Forward primer (10µM) | 2.5 µL |
| Reverse primer (10µM) | 2.5 µL |
| water (Nuclease free) | 18.5 µL |
| Golden Gate Assembly Product | 1.5 µL |
| Total volume | 50 µL |
Table 8. Material required for Q5 PCR after linear Golden Gate Assembly

Methods

1. Add the above recipe into PCR tubes

2. Spin down with a centrifuge

3. Thermocycler Conditions

* 98°C 30 seconds

* Cycles (30 cycles)

+ 98°C 15 seconds

+ Tm 30 seconds (calculated with NEB Tm calculator)

+ 72°C 20 seconds/kB

* 72°C 5 minutes
* 4°C hold

4. After PCR, transfer 5μL for gel electrophoresis to check the band size (Refer to the gel electrophoresis protocol)

**DNA purification**

Refer to the [PCR product purification protocol](https://2022.igem.wiki/cambridge/experiments#purification)

### Making glycerol stocks

Purpose: Create stocks of bacteria with desired plasmid for long-term storage

Material

Cyro tubes, 50% glycerol, bacterial culture

Methods

1. 500μL of culture is added to 500μL of 50% glycerol in cyro tube

* Note: make 2 glycerol stocks per sample

2. Pipette up and down the mixture to mix

3. Put the glycerol stocks into the -80°C freezer

### Miniprep

Purpose: To extract plasmid DNA from bacteria

Material

ThermoFischer MiniPrep Kit, 1.5mL tubes

Methods
All centrifuge steps are carried out at 17000g

1. Transfer all the remaining culture from snapcaps into 15mL falcon tubes

2. Centrifuge at 5000rpm for 5 minutes

3. Discard all supernatent
4. Add 250μL of Resuspension buffer into the tube and vortex until well mixed

5. Transfer all the solution into a 1.5mL tube
6. Add 250μL of Lysis solution and invert 4-6 times (leave for 1-3 minutes)

* Do not wait for more than 5 minutes

7. Add 350μL of Neutralisation solution and invert 4-6 times and wait for 5 minutes

8. Centrifuge for 10 minutes
9. Transfer the supernatent into the spin column, ensure not to take any pallet. If the pallet becomes dislodged, repeat the centrifuge step

10. Centrifuge for 1 minute
11. Wash with 500μL wash solution and wait for 5 minutes, centrifuge for 1 minutes and discard flow through

12. Repeat step 11
13. Transfer the column into a new empty 1.5 mL tube then centrifuge for 10 minutes to remove remaining wash solution

14. Transfer the spin column into a new 1.5 mL tube
15. Add 36μL of warmed elution buffer and incubate for 2 minutes
16. Centrifuge for 2 minutes to elute the samples

17. Measure the yield and purity on Nanodrop

### Oligo Annealing

Purpose: to anneal 2 single strand complementary DNA together into double stranded

Material

Primers for oligo annealing (100μM), water, Thermocycler, PCR tubes

Methods

1. Take 1μL of each primer and add it into 8μL of water into PCR tubes

2. Spin down with a centrifuge

3. Thermocycler protocol

* 95°C 2 minutes

* -1°C/min until 10°C

* Hold at 4°C indefinitely

4. [PCR product purification](https://2022.igem.wiki/cambridge/experiments#purification)

### Q5 PCR

Purpose: to amplify template DNA with minimal error rate

Materials
Q5 PCR Hot Start Master Mix, forward primer, reverse primer, nuclease free water, template DNA, PCR tube, thermocycler

| Q5 PCR amplication contents | 1X (25µL) |
| --- | --- |
| 2X Q5 Hot Start Master Mix | 12.5 µL |
| Forward primer (10µM) | 1.25 µL |
| Reverse primer (10µM) | 1.25 µL |
| water (Nuclease free) | 9 µL |
| Template DNA | 1 µL |
| Total volume | 25 µL |
Table 9. Material required for 25µL Q5 PCR reaction

Methods

1. Add the above recipe into PCR tubes

2. Spin down with a centrifuge

3. Thermocycler Conditions

* 98°C 60 seconds

* Cycles (35 cycles)

+ 98°C 15 seconds

+ Tm 15 seconds (calculated with NEB Tm calculator)

+ 72°C 20 seconds/kB

* 72°C 2 minutes
* 4°C hold

### PCR purification

Purpose: To purify and concentrate DNA Product after PCR

Material

DNA Clean & Concentrator (Zymogen), 1.5mL tubes

Methods

All centrifugation steps should be performed 16,000 x g.

1. Add the 5 volumes of DNA Binding Buffer to each volume of DNA sample. Mix briefly by vortexing.

2. Transfer the mixture to a spin column with a collection tube

3. Centrifuge for 30 seconds, discard flow-through

4. Add 200 µl DNA Wash Buffer into the column and centrifuge for 30 seconds, discard flow through

5. Repeat step 4

6. Add 10 µl Elution Buffer into the column and incubate for 2 minutes

7. Transfer the column into a 1.5mL tube and centrifuge for 30 seconds to elute the DNA
8. Use nanodrop to measure the concentration of DNA product

### Making LB agar selection plates

Purpose: To make selection plates for plating of bacteria

Material

500mL bottle, LB agar, water, autoclave, petri dishes, Biological Safety Cabinet

Methods

1. Add 16g of LB Agar Miller into 400 mL of deionised water into the 500 mL bottle

* never fill the whole 500 mL bottle completely

2. Shake the bottle until well-mixed

3. Autoclave the bottle

* Loosen the bottle lid
* Stick Autoclave tape onto the bottle

4. After autoclaving, take the bottle out out of the autoclave and allow it to cool
5. In the Biological Safety Cabinet, add in 1 in 1000 dilution of 1000X appropriate antibiotics if necessary
6. Pour about 20-25 mL of LB agar into the petri dishes
7. Wait for the agar to solidy and store it in the 4°C fridge

### Making LB broth medium

Purpose: To culture bacteria overnight

Material

500mL bottle, LB broth, water, autoclave

Methods

1. Add 10g of LB Broth Miller into 400 mL of deionised water into the 500 mL bottle

* never fill the whole 500 mL bottle completely

2. Shake the bottle until well-mixed

3. Autoclave the bottle

* Loosen the bottle lid
* Stick Autoclave tape onto the bottle

4. After autoclaving, take the bottle out out of the autoclave and allow it to cool
5. Store the bottle in room temperature

[New Parts](#parts)

[P\_BAD promoters](#pbad)

[Autoregulatory negative feedback loop](#neg)

[Downstream site insertion](#dss)

StayGold GFP characterisation ▼

[Purpose](#intro)

[Design](#design)

[Build](#build)

[Test](#test)

[Experiment 1](#exp1)

[Results (Experiment 1)](#result1)

[Experiment 2](#exp2)

[Results (Experiment 2)](#result2)

[Troubleshooting](#troubleshoot)

[References](#ref)

Results

---

In this page, we showcase the achievements we have made in the project. The following are the list of achievements that we have made throughout the project!

## Cloning new Level 0 and Level 1 Parts

In our project, our goal is to clone level 0, 1 and 2 parts to build an antithetical integral controller. As we are utilising [JUMP assembly](https://2022.igem.wiki/cambridge/design#jump), we have created 39 Level 0 and 37 Level 1 parts that will be a part of the antithetic integral controller. To learn more about the parts that we have the created, please refer to the Level 1 table in the [Parts](https://2022.igem.wiki/cambridge/parts) page. Unfortunately, we were not able to build any level 2 circuits (see below). This meant that we were not able to test any open loop circuits and all of our antithetical integral controller circuits that would have been perturbed.

## Characterisation of P\_BAD promoters

araC is one of the feedback species being utilised for our circuit, characterised by Meyer (2019). As we are considering using araC to activate the P\_BAD promoter, we perform part improvement by designing a family of new P\_BAD through optimisation. For more details about the results of P\_BAD part improvement, please refer to the [Part Improvement](https://2022.igem.wiki/cambridge/improve) page.

## Characterisation of the autoregulatory negative feedback loop

Autoregulatory negative feedback is a type of circuit design motif that exists in nature to improve system response. To compare the response of the autoregulatory negative feedback circuit with the antithetic integral cotroller, we characterise the autoregulatory negative feedback in response to perturbation. For more details, please refer to the [Engineering Success](https://2022.igem.wiki/cambridge/engineering#neg) page.

## Downstream site insertion into Level 2 acceptor (pJUMP47-2A)

In order to create a perturbation circuit to test whether the antithetic integral controller can robustly adapt, we require 5 transcription units, thus we need to utilise the downstream site of the JUMP vector. During our project, we successfully clone the perturbation circuit into the downstream site through designing a 5' adapter and a 3' adapter. For more details on the success of the integration of the perturbation circuit into the downstream, please refer to the [Engineering Success](https://2022.igem.wiki/cambridge/engineering#dss) page.

## StayGold GFP Characterisation

### Purpose

As we are using a low copy number plasmid in our main project, we characterise a new fluorescent protein StayGold GFP which has a considerably better excitation coefficient, brightness and quantum yield compared to other GFPs. To test the fluorescence of a StayGold GFP, we built level 1 circuits but unfortunately we did not observe any fluorescence.

### Design

To characterise StayGold GFP, we have decided to use a low-copy number Level 1 acceptor plasmid (pJUMP27-1A) as a backbone where we will make use of both the main cloning site and the downstream cloning site. To read more about how JUMP assembly works, please refer to the [Rationale](https://2022.igem.wiki/cambridge/design#jump) page in the main project.

Our circuit consists of 1 transcription unit in pJUMP27-1A backbone, consisting of PTet\*, RBS, GFP and DT5. For GFP, in order to compare StayGold GFP with other GFPs available (Figure 1), we look through the Open Reporters in the Distribution Kit Plate 2 to find the 2 GFPs (afraGFP, efasGFP) that has the closest excitation and emission wavelength compared to StayGold GFP to allow better comparison. For the RBS, we have decided to characterise StayGold GFP with both B0033 (weak), B0032 (medium) and B0034 (strong) to see the effect (Figure 2). Quantifying the effect of the strength of RBS is important because fluorescent proteins form aggregates when present in high concentrations. DT5 is an ultrastrong double terminator - discussion of the choice and rationale of terminator usage is discussed in the [Design](https://2022.igem.wiki/cambridge/design-detail#seq) page.

![StayGold GFP compared to other GFP](https://static.igem.wiki/teams/4491/wiki/staygold/staygold-gfp.png)

Figure 1. Circuit diagram of comparing StayGold GFP with other GFP available in the Distribution Kit 2

![StayGold GFP with different RBS strengths](https://static.igem.wiki/teams/4491/wiki/staygold/staygold-rbs.png)

Figure 2. Circuit diagram of comparing StayGold GFP with other GFP available in the Distribution Kit 2

The backbone chosen for the characterisation of StayGold GFP is pJUMP27-1A because it is a low copy number plasmid. As discussed in the [Rationale](https://2022.igem.wiki/cambridge/design#DV) page, a lower copy number plasmid both reduce metabolic burden and noise.

The identifier of the constructs used in our characterisation of StayGold GFP in the following Table:

| ID | Promoter | RBS | CDS | Terminator | Purpose |
| --- | --- | --- | --- | --- | --- |
| LVL1 sinI | PTet\* | B0032 | sinI | DT54 | Negative control |
| 41 | PTet\* | B0032 | StayGold GFP | DT5 | Characterisation of StayGold GFP with B0032 |
| 42 | PTet\* | B0033 | StayGold GFP | DT5 | Characterisation of StayGold GFP with B0033 |
| 43 | PTet\* | B0034 | StayGold GFP | DT5 | Characterisation of StayGold GFP with B0034 |
| 45 | PTet\* | B0032 | afraGFP | DT5 | Compare StayGold GFP with afraGFP |
| 45 | PTet\* | B0032 | efasGFP | DT5 | Compare StayGold GFP with efasGFP |
| PB10 | PBadwt | B0032 | StayGold GFP | DT5 | Troubleshoot: Characterise StayGold GFP with a different promoter |
Table 1. ID of the constructs we have created for the characterisation of StayGold GFP

## Build We perform JUMP assembly to clone the transcription unit into pJUMP27-1A using the enzyme BsaI to clone the transcription unit into the main cloning site. For the protocol we used for [JUMP assembly](https://2022.igem.wiki/cambridge/experiments#jump), please refer to the Experiments page. After golden gate assembly, the construct is then chemically transformed into DH5a cloning strain and plated out. For details of the protocol, please refer to the [Chemical transformation](https://2022.igem.wiki/cambridge/experiments#chem_trans) protocol on the Experiments page. After overnight incubation on Kanamycin selection plates, we want to confirm the construct we want exists in the colony. To do this, we perform [colony PCR](https://2022.igem.wiki/cambridge/experiments#cPCR) and [gel electrophoresis](https://2022.igem.wiki/cambridge/experiments#gel) on a non-fluorescent colony that does not contain superfolder GFP and check whether it has the correct length of amplicon after colony PCR (cPCR). For the details of the protocol, please refer to the to the the Experiments page. For the colony with the correct cPCR amplicon length on gel electrophoresis, the colony is being grown overnight and [glycerol stocks](https://2022.igem.wiki/cambridge/experiments#glycerolstocks) and [Miniprep](https://2022.igem.wiki/cambridge/experiments#miniprep) are being done the next day. For details of the protocol, please refer to the Experiments page. Although the existence of a correct band in colony PCR is a good indication that plasmid with the correct construct is present, the length of colony PCR of the correct construct and the control is very similar (both about 1300bp) and thus will be indistinguishable with a gel. Moreover, to make sure that there is no mutation within the construct, the sequence has to be verified through sequencing. Test Before we perform the plate reader assay, during the cloning process, we have already noticed some interesting features of StayGold GFP. For superfolder GFP, it has a high fluorescent intensity that can be seen with the naked eye on selection plates. Therefore, we have expected to be able to see StayGold GFP fluorescing in the selection plate because DH5a does not have tetR within them some Ptet\* should not be repressed, thus StayGold GFP should be expressed. However, the colonies we picked and subsequently for sequence verification are all non-fluorescing. However, this may be because Ptet\* is not a strong enough promoter itself to show fluorescent. Therefore, we believe doing a plate reader assay is the best choice to confirm the fluorescent of StayGold GFP. After sequence verification, we transform the plasmid into the test strain. For this experiment, we are using the Marionette strain from Meyer (2019) where tetR is present in the cell strain. The tetR present in the cell strain represses Ptet\* where the repression will be lifted when the inducer aTc is added. This allows ut to control whether the expression of StayGold GFP is turned on or off. To transform the plasmid into Marionette strain, we have to use electroporation as Marionette strain itself is not competent. The [electroporation protocol](https://2022.igem.wiki/cambridge/experiments#electroporation) can be found on the Experiments page. Experiment 1 To test out how the concentration of inducers will affect the expression of StayGold GFP, we have designed our plate reader assay with different amounts of aTc inducers on several of our different constructs. We have chosen the range of aTc inducers used based on the characterisation done in Meyer (2019) on the Ptet\* promoter. In the experiment, we have put in 2 negative controls (no\_cells, Marionette\_LVL1sinI) and Marionette\_sfGFP as positive control as superfolder GFP is known to fluoresce. The test plasmids we have decided to use is ID41 and ID42 which is a transcription unit consisting of B0032 and B0033 with StayGold GFP respectively. The aim of the experiment is to compare the fluorescent intensity of StayGold GFP with superfolder GFP and to compare the fluorescent intensity of StayGold GFP with different strengths of ribosome binding sites. The plate reader layout is shown in Figure 3. ![StayGold GFP characterisation plate layout 1](https://static.igem.wiki/teams/4491/wiki/staygold/staygold-exp1-layout.png) Figure 3. Plate layout for experiment 1 Results of Experiment 1 Looking at the numerical data of the plate reader experiment. We can see that the fluorescent intensity of the superfolder GFP is a lot higher than that of the constructs containing StayGold GFP. To confirm this, a graph of the fluorescent intensity against time is plotted which is shown in Figure 4. ![StayGold GFP characterisation Experiment 1](https://static.igem.wiki/teams/4491/wiki/staygold/fi-exp1-new2.png) Figure 4. Result from experiment 1 showing the fluorescent intensity of StayGold GFP is much lower than that of superfolder GFP. For detail explanation of constructs, please refer to the [table](#table_construct) above. Experiment 2 (Troubleshooting) During Experiment 1, we did the experiment pipetting by hand which we have learnt is an error prone process. Moreover, as the first experiment shows that StayGold is not fluorescing, we have decided to repeat the experiment to troubleshoot the results. To do this, we have decided to conduct the experiment using both DH5a and the Marionette strain with all the constructs we have for StayGold, efasGFP and afraGFP with Ptet\* with no inducer (aTc) added or with a concentration (2μM) leading to maximal response added. We have also included a construct with the Pbad wt promoter with StayGold GFP to confirm that it is not the promoter that is not working with and without arabinose. Therefore, for this second iteration of the experiment, we have 14 different culture conditions altogether which makes pipetting manually difficult and error prone. As a result, we have decided to make use of automation. We perform the experiment using OT-2 robot which allows automated pipetting with a Python script invented by Camillo Moschner, one of the instructors of the iGEM Team (Moschner *et al.,* 2022). The plate layout is shown in the figure 4, a screenshot of the Python script we used to control the OT2 robot. As we can see, the plate layout is very complex and the experiment is benefiting hugely from automation. The day before the plate reader experiment, we overnight culture the 12 different strains of cells from a streaked out plate into EZRDM for approximately 12 hours. After overnight culture, we pipette 14μL of the respective cell culture into PCR tubes and place it in the order shown in the screenshot of the Python script output into the robot (Figure 4). We also made EZRDM media with an appropriate Kanamycin and inducer concentration into Falcon tubes and place in the appropriate order as shown in the figure. ![StayGold GFP characterisation plate layout 2](https://static.igem.wiki/teams/4491/wiki/staygold/staygold-exp2-layout.png) Figure 5. Plate layout and culture layout of the robot for experiment 2 After preparation, the following plate reader settings were used to characterise StayGold GFP. ![StayGold GFP troubleshoot experiment plate reader settings 1](https://static.igem.wiki/teams/4491/wiki/staygold/221004-igem22-sggfp-trsht-prsetting-sggfp-1.png) Figure 6. Plate reader settings for fluorescent intensities ![StayGold GFP troubleshoot experiment plate reader settings 2](https://static.igem.wiki/teams/4491/wiki/staygold/221004-igem22-sggfp-trsht-prsetting-mvenus-2.png) Figure 7. Plate reader settings for fluorescent intensities ![StayGold GFP troubleshoot experiment plate reader settings 3](https://static.igem.wiki/teams/4491/wiki/staygold/221004-igem22-sggfp-trsht-prsetting-sggfp-3.png) Figure 8. Plate reader settings for fluorescent intensities ![StayGold GFP troubleshoot experiment plate reader settings 4](https://static.igem.wiki/teams/4491/wiki/staygold/221004-igem22-sggfp-trsht-prsetting-sggfp-4.png) Figure 9. Plate reader settings for fluorescent intensities (gain) ![StayGold GFP troubleshoot experiment plate reader settings 5](https://static.igem.wiki/teams/4491/wiki/staygold/221004-igem22-sggfp-trsht-prsetting-od600-2.png) Figure 10. Plate reader settings for OD600 ![StayGold GFP troubleshoot experiment plate reader settings 6](https://static.igem.wiki/teams/4491/wiki/staygold/221004-igem22-sggfp-trsht-prsetting-wizard.png) Figure 11. Plate reader presetting Wizard showing protocol we used for the plate reader assay Results The results again show that StayGold GFP does not fluoresce as expected. The following graphs are plotted as a comparison. We first plotted efasGFP, afraGFP and StayGold GFP (all RBS strengths) on the same plots under the same promoters (Ptet\*) for both the DH5a strain (Figure 10) and the "Marionette" strain (Figure 11). The result shows that inside both Marionette strain (with aTc added) or DH5a, although efasGFP is fluorescing strongly and is saturating the plate reader at this gain, both afraGFP and StayGold GFP did not have a high fluorescence intensity. ![fluorescent intensity of DH5a comparison](https://static.igem.wiki/teams/4491/wiki/staygold/dh5a-41-46-fi-new.png) Figure 12. The fluorescent intensities of constructs (ID41, 42, 43, 45 and 46) in DH5a cells. Notice how the fluorescent intensity of efasGFP is a lot higher than that of StayGold GFP and afraGFP. For detail explanation of constructs, please refer to the [table](#table_construct) above ![fluorescent intensity of Marionette comparison](https://static.igem.wiki/teams/4491/wiki/staygold/marionette-41-46-fi-new.png) Figure 13. The fluorescent intensities of constructs (ID41, 42, 43, 45 and 46) in "Marionette" strain. Notice how the fluorescent intensity of efasGFP is a lot higher than that of StayGold GFP and afraGFP. For detailed explanation of constructs, please refer to the [table](#table_construct) above To ensure aTc is performing what we expect of alleviating the repression of tetR on Ptet\*, the time series of fluorescent intensity of efasGFP inside the Marionette strain with and without aTc is plotted on figure 12, showing that the addition of aTc does increase fluorescent intensity. ![fluorescent intensity of Marionette (ID45) with and without aTc](https://static.igem.wiki/teams/4491/wiki/staygold/marionette-45-atc-fi-new.png) Figure 14. The fluorescent intensities of ID45 in "Marionette" strain with and without aTc added. For detailed explanation of constructs, please refer to the [table](#table_construct) above Apart from comparing the effects with different aTc concentrations added, we also use a Pbad promoter (PB10) to ensure that the previous low fluorescent intensity signal was not due to the Ptet\* promoter. The following figure shows the fluorescent intensity of StayGold GFP under the Pbad wild type promoter with different concentrations of arabinose. As we can see, the fluorescent intensity did not significantly change even with the addition of arabinose. ![fluorescent intensity of Marionette (PB10) with and without arabinose](https://static.igem.wiki/teams/4491/wiki/staygold/marionette-pb10-arabinose-fi-new.png) Figure 15. The fluorescent intensities of PB10 in "Marionette" strain in different arabinose concentration. Notice how the fluorescent intensity does not change significantly despite the addition of arabinose. For detailed explanation of constructs, please refer to the [table](#table_construct) above Plotting the constructs containing StayGold GFP of different RBS strengths on the same graph for both DH5a (Figure 13) and the Marionette strain (Figure 14), we see that although the trace of B0032 and B0033 is very similar, the trace of B0034 is significantly higher. This may imply that putting StayGold GFP under an even stronger promoter may cause it to have a way higher fluorescent intensity. ![fluorescent intensity of DH5a comparison (Different RBS)](https://static.igem.wiki/teams/4491/wiki/staygold/dh5a-41-43-fi-new.png) Figure 16. The fluorescent intensities of constructs (ID41, 42, 43) in "Marionette" strain. Notice how the fluorescent intensity of StayGold GFP with RBS B0034 is higher than that of RBS B0032 and RBS B0033. For detailed explanation of constructs, please refer to the [table](#table_construct) above ![fluorescent intensity of Marionette comparison (Different RBS)](https://static.igem.wiki/teams/4491/wiki/staygold/marionette-41-43-fi-new.png) Figure 17. The fluorescent intensities of constructs (ID41, 42, 43) in "Marionette" strain. Notice how the fluorescent intensity of StayGold GFP with RBS B0034 is higher than that of RBS B0032 and RBS B0033. For detailed explanation of constructs, please refer to the [table](#table_construct) above DBTL cycle: Trouble shooting attempts Throughout building our level 2 to create antithetic integral controllers we encountered a number of challenges in our cloning process. We initially successfully cloned 39 parts into level 0 plasmids and 37 transcriptional units into level 1 plasmids, however we were unable to complete any level 2 system. To overcome these cloning problems we tried implementing a number of troubleshooting techniques. Success rate of the cloning process and optimisation strategies Cloning our DNA parts into the level 0 plasmids was relatively straightforward however making and cloning the level 1 parts was substantially harder. The average overall success rate was about 25%. Once Golden Gate Assembled (GGA) and transformed, approximately 30% of the resulting colonies failed to show any non-fluorescent colonies (those which had inserted into backbone superfolder GFP (sfGFP) CDS) indicating that the golden gate protocol failed. The success rate within a given successful golden gate assembly to transformation varied dramatically from 10 to 90%. We attempted to optimise this by test out different cycle numbers and the time in each cycle (see overnight compared to same day GGA in protocols). However, little difference in golden gate assembly success occurred. From those non-fluorescent colonies about 50% had correct cPCR amplicon length. Often we accidently picked sfGFP colonies that were not fluorescing brightly. From these colonies, typically 2-3 were sequenced. Often the sequencing results showed that the transcriptional units were missing parts. Often these were RBS or promoters which were too small to distinguish on a cPCR gel given that they could be smaller than 100 bp. Module 1B with Anderson promoter This was particularly a problem for module 1 B’s with Anderson promoters. Our design for our antithetic integral controller circuit involves an Anderson promoter constitutively expressing Z1 in Level 1 Module B. Without these circuits we couldn’t build our level 2 circuits. When we are trying to clone in the transcription unit into Level 1 Module B, the promoter and RBS are removed from the assembly such that only the CDS and the Terminator are present. This has happened consistently throughout the cloning process where out of the 20+ tries for various different types of promoters and RBS, only 2 of the assemblies are successful with the whole transcription unit being cloned in. In all cases, both the RBS and promoter were missing. ![Missing Anderson Promoter and RBS](https://static.igem.wiki/teams/4491/wiki/results/level1-moduleb.png) Figure 18. Incorrect golden gate assembly of module C and D but not A and B. This occurred most often for Anderson promoters and module B ![Missing Anderson Promoter and RBS](https://static.igem.wiki/teams/4491/wiki/results/level1-moduleb-zoom.png) Figure 19. Zoomed in image of Figure 18 We find this strange given JUMP assembly has ensured that the overhang of CDS is not compatible with the overhang of the backbone thus CDS and the backbone cannot be stitched together. In silico cloning in Snapgene further suggested that the backbone should not be able to be stitched directly with the CDS and terminator. We have also found that the transformation efficiency of these plasmids into cells are very low where almost all of the cells on plates contains superfolder GFP, suggesting that cloning was unsuccessful. When we are trying to pick the non-fluorescent colonies, in a lot of the cases, we have resulted in a liquid culture that does not grow in Kanamycin culture and have no bands when running gel electrophoresis after colony PCR. This means that these non-fluorescent colonies do not contain the cloning product we want as well. Typically this indicates that the antibiotic selection has failed but this was controlled for by a negative/positive controls as well as replacing the antibiotics stocks. A variety of strategies were used to tackle this problems. Firstly, we decided to try double and then quadruple the concentration of promoter and RBS to see whether the situation has improved. However, this yields the same result. To test whether it was a problem with module B, we cloned Pbad\_wt for our characterisation circuit. Out of the three we tried, two worked. However the success of these cloning reactions remained low. We further hypothesised that since we were using strong constitutive promoters could have resulted in metabolic toxicity. One attempt to mitigate this was to grow the plates at 30C for 48 hrs rather then 37C for 12 hrs which should reduce the toxicity. However, this resulted in little additional success. Furthermore, even weak Anderson promoters (J23105) and weak RBS (B0033), in which high toxicity/metabolic load were not to be expected, also failed. A final strategy to make the level 1 parts was to use a linear GGA protocol instead of plasmid GGA. We had to design overhang adapter sequences with the appropriate level 1 restriction sites. The parts were then assembled using linear GGA, PCR amplified and purified. We identified bands of correct size but were unable to fully assemble the level 1 plasmid. ![Gel electrophoresis](https://static.igem.wiki/teams/4491/wiki/staygold/gel-cropped.jpeg) Figure 20. Successfully golden gated module B with Anderson promoters. Level 1 ID11 and 30. Level 2 cloning difficulties Similar wto the level 1 circuits we were only ever to assemble two out of the four level 1 transcriptional units into a level 2 system. We tried approximately 20 cloning runs, trialling different golden gate protocols and restriction enzymes (Bsmb1 and Esp1) but all failed. In all cases only module A and B were found and attached unexplainably directly to the backbone. ![Level 2 difficulties](https://static.igem.wiki/teams/4491/wiki/results/level2-diff.png) Figure 20. Level 2 golden gate assembly, with module C and D level 1 transcriptional units missing. References 1. Meyer, A.J., Segall-Shapiro, T.H., Glassey, E. et al. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nat Chem Biol 15, 196–204 (2019). https://doi.org/10.1038/s41589-018-0168-3 2. Marcos Valenzuela-Ortega, Christopher French, Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology, Synthetic Biology, Volume 6, Issue 1, 2021, ysab003, https://doi.org/10.1093/synbio/ysab003 3. Hirano, M., Ando, R., Shimozono, S. et al. A highly photostable and bright green fluorescent protein. Nat Biotechnol 40, 1132–1142 (2022). https://doi.org/10.1038/s41587-022-01278-2 4. Moschner Camillo, Wedd Charlie, Hardo Georgeos, Bakshi Somenath (2022). The iBioFoundry: Automated, Low-Cost, High-Throughput Experimentation (IWBDA manuscript accepted)