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Part Mining

The goal of our project is to show robust perfect adaptation by building an antithetic integral controller. To achieve this, we have to consider the following

1. Destination vector


2. Reporter


3. Transcription factor


4. Characterisation

Destination Vector

The circuit we use for characterisation of our circuit requires a low noise and burden because building a multi-transcription unit circuit is very complex. Through doing the characterisation for Bronze Contribution, we have figured out that utilising a low copy number plasmid allows us to lower metabolic burden and reduce noise, giving us more reliable results. It may seem counterintuitive that a lower copy number plasmid yields lower noise as a lower copy number intuitively lead to higher plasmid number fluctuations. However, low copy number plasmid has lower noise because there is a feedback system in place to maintain the copy number of the plasmid in the cell. In a high copy number plasmid, the feedback system is removed thus counter intuitively leading to higher noise in plasmid copy number.

Reporter

As our characterisation is in a low copy number plasmid, we require a reporter that has a high intensity so it can be easily detected. Through literature search, we have found a new GFP called StayGold GFP from Hirano (2022) that has improved characteristics compared to other GFP reporters. The details of the characterisation can be found in the StayGold GFP characterisation page.

In order to measure the amount of output we have from the antithetic integral controller circuit, we have decided to build an operon that put the reporter and the output under the same promoter. As JUMP assembly requires us to clone in the promoter, RBS, CDS and terminator at the same time and does not allow polycistronic assemblies as is this case, we have ordered the RBS, mVenus and terminator as its own part called MegaT and use the whole part as the terminator. For more information about the design of MegaT, please refer to the MegaT characterisation part of the Engineering Success page.

Transcription factor

One of the feedback species we considered using for our circuit is araC that was characterised in Meyer (2019) and was the feedback species used in Aoki (2019) for their design of implementing the antithetic integral controller in bacteria. In order to improve ther response of araC on the Pbad promoter. We have decided to characterise Pbad and perform part improvement on the Pbad promoter. For more details of the part improvement of the Pbad promoter, please refer to the Part Improvement page.

Characterisation

As the design of the antithetic integral controller requires the use of controller species Z1 and Z2 where they annihilate with each other to form the error signal of the circuit, characterising these parts becomes important. As a result, we have designed the characterisation plasmid to characterise the controller species (Figure 1). For more details, please refer to the Design page.

Design

To show that the antithetic integral controller show RPA, we have designed the antithetic integral controller, with a perturbation circuit for testing. To show that this performs better than other adaptation strategies, we are comparing the antithetic integral controller with an autoregulatory negative feedback loop and an open loop circuit. The design of the circuit is shown below.

Antithetic

The antithetic integral controller circuit (Figure 2) is mathematically proven to be able to robustly adapt. To read more about the description of the circuit design, please refer to the Design page. To perturb the circuit, we have also integrated part of the perturbation circuit into the downstream site. To read more about the design of the downstream site, please refer to the downstream site integration part of the Engineering Success page.

Negative Feedback

Negative feedback is a type of circuit design motif that exists in nature to improve system response. We aim to compare the antithetic integral controller motif with the autoregulatory negative feedback motif (Figure 3) to see how they respond to perturbation. To read more about how we have characterised the negative feedback circuit, please refer to the negative feedback circuit in the Engineering Success page.

Open Loop

To show adaptation strategies, we have designed an open loop circuit (Figure 4) to compare with in response to perturbation. To read more about the design of the open loop circuit, please refer to the Design page

Implementation

To implement the characterisation of the circuits we have designed, we require various different equipments. We have access to the plate reader, microfluidic device, microscope and automation.

As we require a fully factorial experimental design for the characterisation of the PBad promoter which goes far beyond the capacity of the 96 well plate. We have decided to use automation provided by the lab from one of our instructors to perform the experiment with a 384 well plate, allowing us to identify which factors (DNA sequence and inducer concentration) is important with a high throughput. For the details of the automation and characterisation, please refer to the Part Improvement page.

Joint Universal Modular Plasmid (JUMP) Assembly

Joint Universal Modular Plasmids (JUMP) is a new toolkit allowing efficient golden gate cloning. The distribution kit parts come in vectors with a flanking sequence compatible to the JUMP format. For the parts not in the distribution kit, we have to put the flanking JUMP compatible sequence onto the sequence that we are ordering. After which, they are cloned into the level 0 vector (pJUMP18_Uac) using the enzyme BsmbI or Esp3I. After this, we proceed on cloning it into level 1 vector. For level 1 JUMP vectors, there are module A, module B, module C and module D. Within each module we aim to clone inthe promoter, RBS, CDS and terminator. To do this, the 5’ and 3’ sequence have to be JUMP compatible. Table 1 shows the flanking sequence required for each part. Notice that for promoter and RBS, the sequence is the same as CIDAR MoClo while it isn’t the case for CDS and terminator. The appropriate parts are cloned into module A, B, C and D respectively with the enzyme BsaI.

Table 1. the flanking sequence required for each part for CIDAR MoClo and JUMP assembly.

	Module A (5')	Module A (3')/ Module B (5')	Module B (3')/ Module C (5')	Module C (3')/ Module D (5')	Module D (3')
Flanking sequence	GGAG	AATG	AGCC	TTCG	CGCT

Module A, B, C and D each has a compatible flanking sequence on the 5’ and 3’ end to allow cloning into the level 2 acceptor. The flanking sequences of Module A, B, C and D respectively are shown in Table 2. The 4 modules are cloned into Level 2 acceptor using BsmbI/Esp3I.

Table 2. The flanking sequences of Module A, B, C and D.

	Module A (5')	Module A (3')/ Module B (5')	Module B (3')/ Module C (5')	Module C (3')/ Module D (5')	Module D (3')
Flanking sequence	GGAG	AATG	AGCC	TTCG	CGCT

The plasmid at each level have a different antibiotic selection marker. Level 0 requires ampicillin, level 1 requires Kanamycin and level 2 requires Spectinomycin.
Each JUMP vector also consists of an upstream and a downstream site where extra transcription units can be cloned in. To clone into upstream or downstream site, a linearly ligated transcription unit with the appropriate restriction enzyme recognition site on both 5’ and 3’ end of the transcription unit is needed. To clone into the upstream site, the enzyme AarI is required while for the downstream site, the enzyme BbsI is required.

References:

1. 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


2. Aoki SK, Lillacci G, Gupta A, Baumschlager A, Schweingruber D, Khammash M. A universal biomolecular integral feedback controller for robust perfect adaptation. Nature. 2019 Jun;570(7762):533-537. doi: 10.1038/s41586-019-1321-1. Epub 2019 Jun 19. PMID: 31217585.


3. Rhodius VA, Segall-Shapiro TH, Sharon BD, Ghodasara A, Orlova E, Tabakh H, Burkhardt DH, Clancy K, Peterson TC, Gross CA, Voigt CA. Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters. Mol Syst Biol. 2013 Oct 29;9:702. doi: 10.1038/msb.2013.58. PMID: 24169405; PMCID: PMC3817407.


4. Newman JA, Rodrigues C, Lewis RJ. Molecular basis of the activity of SinR protein, the master regulator of biofilm formation in Bacillus subtilis. J Biol Chem. 2013 Apr 12;288(15):10766-78. doi: 10.1074/jbc.M113.455592. Epub 2013 Feb 21. PMID: 23430750; PMCID: PMC3624457.


5. Meyer AJ, Segall-Shapiro TH, Glassey E, Zhang J, Voigt CA. Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors. Nat Chem Biol. 2019 Feb;15(2):196-204. doi: 10.1038/s41589-018-0168-3. Epub 2018 Nov 26. PMID: 30478458.


6. Park Y, Espah Borujeni A, Gorochowski TE, Shin J, Voigt CA. Precision design of stable genetic circuits carried in highly-insulated E. coli genomic landing pads. Mol Syst Biol. 2020 Aug;16(8):e9584. doi: 10.15252/msb.20209584. PMID: 32812710; PMCID: PMC7436927.