Team:HKU-Hong Kong/Project

Several ideas on the subject of this year's investigation were come up during various brainstorming sessions. Our team picked out the idea of engineering a mechanism that can act as a safety net to prevent genetically modified bacteria from performing undesired tasks in wrong environments.

Bacteria are genetically engineered to perform various functions.Prospective functions include biodegradation of crude oil and killing of cancer cells. Yet, undesired tasks might be performed by the baterium itself as well. The idea of a "bio-safety" net would serve the purpose.

For example, after fulfillment of intended functions or when bacteria have escaped from the intented work zone.

Overall project

ways to achieve this

• Insertion of killing genes
• Regulated by specific promoters

• Promoters respond to changes in the environment

Hence bacteria can and can only function well and survive in a specific environment

Project Details

Killing Mechanism

• Enterobacteriophage T4 Lysis Device
  • We found the biobrick form Berkeley's team from iGEM 2008 a possible way to attain cell destruction.

Uses genes from enterobacteriaphage T4 (endolysin – lysozyme, holin and antiholin). Various promoters can be installed into this device to control lysis. Antiholin has its own constitutive promoter to prevent formation of holin multimers from basal expression. When device is off, higher expression of antiholin is needed for better stability of the device.

• Others
  • Lytic enzyme systems from bacteriophage
    • Lambda
    • phiX174
    • MS2
    • Qβ
    • T2
  • Hybrid designer lytic enzyme

Regulation

Placement

Changes in our plan

Difficulties

• Oil is a mixture of chemicals
• Does not induce promoters
• Difficult to target a specific component in oil
• Dfficult to implement the oil degrading system into E. Coli within the time period available to our project

The changes

After identification of the difficulties, we decided to change our plan and instead of focusing on oil, we will use lactose to imitate ‘oil’. This lactose analogue can also be substituted with different subtracts with appropriate modification on the biobricks in initiating the mechanism. Lactose is used as the analogue owing to various reasons:

• Lactose operon is widely studied
• No reporter needed
• Lac-Z assay
• Optimal difficulty and achievable for 2 months’ time

So, now our plan has changed from making an oil digesting bacterium to a lactose digesting bacterium. After the digestion is done, the bacteria will kill themselves.

The plasmid design

• placBAD: a promoter inducible by lactose or arabinose
• cI: the inhibitor of the pR promoter
• Lac I: The inhibitor of the lac operon
• Lac Z: Beta-galactosidase
• Lac A : lactose acetylase
• Lac Y : lactose permease
• pL : a promoter that can be inhibited by cI

Scenarios

• In the presence of lactose, the lac operon and cI which was originally suppressed by repressor turned on. Transcription, translation and modifications are carried out to express the gene. cI protein and the β-galactosidase which is the hydrolytic enzyme that catalyzes the hydrolysis of lactose were produced. The lactose started to be digested. cI protein is the repressor of the promoter of suicide gene E, pL. The expression of suicide gene was then suppressed.

• The lactose in the cell culture was continuously digested. When all the lactose was digested, the promoter of cI gene and lac operon was suppressed. Noβ-galactozidase and cI protein were produced. In the absence of cI protein, the suicide gene turned on. The expression of gene E (production of E protein) leads to formation of holes in the cell membrane which causes the cell death. The mechanism can be referred to the previous part.

• During the culture of the bacteria, arabinose is added to the LB agar which is the growing medium of the bacteria. The promoter of cI and lac operon can be activated by arabinose. Therefore, cI protein was produced continuously to suppress the expression of the suicide gene. This can prevent the bacterial cell death under un-want conditions.

Possible flaws

• Leaky expression
• The suicide genes won’t work
• The time it takes for cell death

Results

Possible further research