Part:BBa_K5308005

Module environment sensing kill switch

This device is an Cre regulated kill swtich. It solves the dilemma in designing kill switches for enviroment-related project by changing the expression of killred, which could kill bacteria when it's accidently released into environment by daily sunlight.

README

This device is just proof of concept. The "key to sense environment" is of high modularity.

In this part, the expression of Cre is controlled by VanR and vanillic acid. In practical application, we can regulate Cre by other inducible promoters, such as UV in space, salt in Saline-alkali land, low-oxygen underground and anything relative to a specific environment.

Why this part is important

Tackling problems in the environment is an important topic in synthetic biology. However, designing a suitable kill switch is challenging. We usually need to improve chassis' resistance to a specific environment which is also related to its problem. For expamle, if we want to use bacteria to degrade chlorite in the soil, we need to increase its tolerance to chlorite. But if this engineered strain is released to the environment in our daily life, it would have a higher fitness and pertub local environment. On the other hand, we finally need to release it to a specific environment.

So there is a dilemma in designing kill switches for enviroment-related project.

Usage and Biology

Our new design includes using KillRed as a suicide switch, which releases reactive oxygen species to eliminate bacteria in a normal environment. We employ the Cre-loxP system to regulate KillRed's expression. Cre is a cyclization recombinase that interacts with loxP sites to reverse the DNA sequence between them when the sites are inverted. After incorporating a specific "sensor"—an inducible promoter triggered only in the intended environment, such as high salt concentration or low oxygen levels. This controls Cre, with the inducible promoter (Pinducible) determined by the environmental factors relevant to the application.

So when we put the strain in an environment that needs to be improved, Cre will flip the promoter upstream of KillRed, so that Killred will not be expressed and the bacteria will not be killed by sunlight. At the same time, the promoter was flipped, and the expression of population control device (BBa_K3893030) was turned on to control the population number of bacteria.

Part engineering and DBTL cycle

The development of this part was performed by careffuly considerring porential transcription and translation in two stages with helps from exciting software Design of a module environment kill switch. Here, we describe the different steps and the results obtained.

Design

When applying engineered bacteria into the environment for treatment, a safety system is essential to confine the bacteria to their intended environment but not accidentally release and prevent them from affecting the normal microbial community after treatment.

To promise engineered bacteria would be killed after accident release, we choose killred which releases reactive oxygen species to eliminate bacteria under sunlight. The promoter and RBS of killred are set between two loxp sites which can be inverted by Cre.

To promise engineered bacteria will not be kill under specific environment we want to improve, the expression of Cre are controlled be a inducible promoter sensing to in the intended environment, such as high salt concentration or low oxygen levels. Here we used vanillic acid as a proof of concept.

Analysize DNA sequence by de novo DNA

While it depends on the cooperation of terminator, promoter and RBS, we needed to design it carefully by analyzing the expression of KillRed. Here, we used de novo DNA to predict both the transcription rate and translation rate of killRed, and to design the stage changer.

We first set RBS (BBa_B0034) between the downstream loxp and KillRed (A), and after recombining by inducible Cre, the DNA sequence will change (B).

We predicted the potential transcription sites. To our surprise, we found there is a high transcription value, reaching 2416 (A). We noticed that sequence in loxp comprises potential -10 box and -35 box in predicted promoter in yellow (B).

So next we redesigned this cluster. Firstly, we move RBS (BBa_B0034) upstream to loxp, and removed the 'ATG' in Killred. Then, we set a 'ATG' unpstream loxp, and designed a linker between peptide translated from loxp region and killred region (A). After recombining by inducible Cre, bothe RBS and start codon 'ATG' will be inverted, so killred will not be expressed (B). There is also no 'TTG' and 'GTG' for potential weak translation of killRed (B).

Build and Test

We built this part by Gibson, and used sanger sequencing to check successful assembly.

Then we test it in solid LB medium in a light incubator with different concentrations of vanillic acid.

Materials and Methods

1. All experimental measurements were taken with Biotek CytationTM 3, using a 96-well plate. The experimental conditions used in this work are in the Table below
2. The cells were E. coli DH5-alpha transformed with the corresponding plasmid.
3. For all experiments, we worked with an isolated colony that contains the corresponding plasmid.
4. Cultures of 5mL falcon tubes with LB medium with the corresponding antibiotic were prepared and incubated overnight at 37°C, 220 rpm.
5. The overnight cultures were refreshed by inoculating 50 μl into 5 mL of BL with the corresponding antibiotic and incubated at 37°C, 220 rpm for 2 hours.
6. Cultures were diluted 100000x and 50 μl of mixtures were plated into solid LB midium with the corresponding antibiotic with and w/o 200 μM vanillic acid. These plates were incubated 18h in a light incubator.
7. We counted the colony formation units of each plate.

Learn

This part undoubtedly works. In high level of vanillic acid, lots of colonies grew in plates, while when no vanillic acid was added, the colony formation significantly decreased. Remember that vanillic acid is just a proof of concept, it can be set to any other things in specific environment such as high salt concentration or low oxygen levels

Future improvement plan

The inversion of Cre is reversible, so after induced by vanillic acid or other inducer in the environment, it remeans at least 50% expression of killred. This will kill engineered bacteria even if we release it in correct environment.

We will try to change it to PhiC31, Bxb1 and other serine recomnibases. The inversion of attB and attP is irreversible.

Furthermore, we planned to put a key of Population control device (BBa_K3893030, QS-based lysis protein oscilator), this will regulate engineered bacteria with limited population and promising function.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 18
  Illegal NheI site found at 2494
  Illegal NheI site found at 2517
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1766
  Illegal BamHI site found at 4
  Illegal BamHI site found at 42
  Illegal BamHI site found at 507
  Illegal BamHI site found at 2121
  Illegal XhoI site found at 2156
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2272
  Illegal AgeI site found at 193
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2742
  Illegal BsaI.rc site found at 3033