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From 2011.igem.org

5/30 - Research topic selected

Group 1

1. Hydrocarbon absorption

• Because it is hydrophobic, it will not be difficult for it to diffuse through the membrane and the cell wall.
• Other teams for example: used AlnA ptn to increase alkane solubility.
• AlkB2 and ladA were used as transporters. We can try to knock-out those genes that enable microorganisms to degrade alcohol to keep the alcohol stored within them.

Suggestion 1) HAE1 gene, AcrAB genes are an option

Suggestion 2) Increasing absorption rate: increase metabolic rate to speed up the rate at which alkane is degraded.

Suggestion 3) Instead of looking for a suitable transporter, focus on increasing efficiency in synthesizing and extracting wax.

2. Determine the types of enzymes in E.coli

3. Study about alkane monooxygenase

4. Using and synthesizing of wax

• Directly injecting substrate is meaningless. Find out if we can use various substrates.
• Acyl-CoA reductase(AcrM) is the key enzyme.

5. Increasing efficiency

• Overexpression of the gene AcrM
• Modification of inclusion body

Group 2

1. Microorganism that synthesizes biosurfactants and degrades hydrocarbons

• Detection of soil contaminants; Because soil condition is difficult to determine through naked eyes, detection is important. (Removal of contaminant and thereby facilitating soil’s recovery)
• Absorbing the contaminant
• Surfactant expression: Green Fluorescent Protein; House-keeping gene expression: Red Fluorescent Protein
• Because many species of bacteria can synthesize biosurfactant, it may be possible to degrade various contaminants.
• Reason for TU Delft’s unsuccessful experiment: injecting too many genes all at once interferes with the regulation system, resulting in non-favorable results.

- Should we insert the genes all in one cell?

• Heterogeneous genes, if they are expressed at the same time, may cluster. Furthermore, the organism will not be able to sustain and survive a modification of its membrane.
• Facilitation mechanism: Incorporation of a regulation system into TU Delft's mechanism. Insertion of nitrogen fixation gene to bring about a more fertile soil. However, a downside to this method is that a plant must be nearby. Searching for a microorganism that is independent of plants may be an option.
• Nitrogen fixation is not easy. Hydrogenase is used in Nitrogen fixation, but its enzymatic activity is greatly diminished upon exposure to oxygen, thus decreasing efficiency.

- Groups 1 and 2 should combine and study more about Delft.

- We should not be satisfied with simple detection. We must proceed further.

- Advisor will let us know of the possibilities of our experiment.

- Find out why Delft used the genes they used.

- Aiming for bioenergy production

• Substrate selection
• Fermentation by anaerobic bacteria of burnt waste matter from cities is too big of a scale
• Wax seems to be the ideal substrate.

Group 3

1. Auto-inducible and auto-lytic system using quorum sensing

• Quorum sensing gene: amplified with PCR(a PCR that increases rate of mutation)
• Addition of Magnesium ion to a PCR reactant mixture(error-prone condition)
• As PCR proceeds, gene mutations occur and these genes are replicated. Affinity difference between inducer and promoter becomes clearer and the concentration at which transcription begins changes. By screening the replicated genes’ affinity, we can obtain a library, and by inserting a GFP gene near a promoter, degree of expression can be determined. Differentiate genes based on time of GFP expression, measure their affinity, and select desired bacterial species.
• Testing: Upon contacting the desired cell OD, is the target gene expressed? (Inducers differ in the concentrations they recognize.)

2. Basic tool: wax synthesis

• Should the wax synthesis directed toward the cell interior, exterior, within the media?

- Directing it towards the surface of the media will be the most convenient method. Following synthesis, wax will float on the media which we simply will pick up.

• Involvement of wax esterase and EtOH?

- The problem is, wax esterase is a dimer, making it difficult to display. On the other hand, making the microorganism secrete FA is too complex a task. We then came up with the idea of flipping the FA transporter. If that’s difficult, we can try storing and stacking wax as layers within the organism, or try producing alcohol.

• Instead of supplying substrates manually, inducing the microorganism to synthesize its necessary compounds may be possible. In doing so, we should not interfere with the beta-oxidation pathway. Altering such crucial pathway may result in cell death.
• Using Atfat is an option. (Important to note that the type of esterase used depends on whether target FAs are short or long)

3. FA absorption mechanism

• Anaerobic Acinetobacter growth rate is too slow. Desired quantities of products are not synthesized.
• Acinetobacter transport components may be applied to E.coli. May be useful if possible.