Results

System Working Under Real World Conditions.

Synthetic biology aims at using biological part to construct different kinds of devices to realize different functions in real world. Experiments in the laboratory is idealized so it cannot represent the real world conditions all the time. In order to simulate the real world condition without taking the biological materials outside the lab, we can use PET products bought from the regular shop instead of the chemical shop. Considering the organisms are living with each other in real world, we applied the microbial consortium to simulate the real world condition as much as possible.

Using the Real World Substrate

Our project this year is about biodegradation of the widely used palstic PET. As a polymer, PET is hard to degrade and the degrading process is time-consuming. Due to the mild condition of the biodegradation reaction and the restriction of growing of organisms, it is even slower although it is more environmental friendly. Therefore, we use the substitute pNPA (p-nitrophenyl acetate), a kind of small molecular ester as the reagent of enzymatic reaction. However, pNPA is not what we really want to degrade, so we need to measure the PET degrading effects of our enzyme. Actually, pNPA is mainly used to screen for the mutated PETase gene because of the high requirement of efficiency of this step. When we obtain the mutants we want, we used them to degrade PET. In order to simulate the real world condition as much as possible, we used the PET film bought from the online plastic products shop instead of the test reagent bought from the chemical company. The film is originally used as packing material. We soak the cut film into the 75% ethanol solution and wash them with double distilled water before degrading experiments.

Using the Real World Simulated Microbial Consortium

The inspiration of the idea of microbial consortium comes from nature. Actually, bacteria never exist alone in our nature, they co-work and cooperate together to achieve an aim or live better in a special condition. Thinking from this point, we established a special bacteria consortium for this enzyme catalysis reaction in order to not only improve the degradation effect, but also simulate the real world condition. In this system ,we applied three kinds of organisms, Pseudomonas putida KT2440, Rhodococcus jostii RHA1 and Bacillus stubtilis 168 (or Bacillus stubtilis DB 104), to degrade the PET and the harmful product EG and TPA to carbon dioxide or generate biodegradable plastic PHA. We also decided to introduce the Cyanobacterial into the system in the future in order to make this system use only solar energy to work.

Experiment Data of Respective Section

Protein Engineering

xxx

Microbial Consortium

xxx

R-R system

1.Results of inclusion body based reporting system.

The red fluorescence can be observed by bare eyes. We set up four groups:
A. No E.coli.
B. E.coli with empty plasmid pUC19.
C. E.coli with pUC19+CpxR-RFP.
D. E.coli with pUC19+CpxR-RFP+PETase gene.
The result is as the following picture. (After centrifugation with the speed of 12000rpm for 1min)

Fig.1. Directed observed fluorescence of inclusion body based reporting system. (From left to right: A, B, C, D)

We found that the group with part BBa_K339007 and PETase gene showed deepest red in all of the four groups. The group with only part BBa_K339007 also showed a little red, we speculate that this might be caused by the basic expression of some genes in E.coli and some of the expression products formed inclusion bodies. It might also be caused by the basic expression of the RFP gene because the CpxR promoter might also, though not so strongly, start the transcription even without the induction of CpxR protein. The group with empty plasimd did not showed any red color and the group with no E.coli cultured did not have any sediment at the bottom of tube.

2.Results of inclusion body based cell lysis regulation system.

We first did a experiment to measure the cell lysis effect of ddpX gene. We simply use the IPTG inducible T7 promoter to regulate the expression of ddpX gene. We totally set four groups:
1. E.coli wildtype.
2. E.coli wildtype added IPTG.
3. E.coli with ddpX gene, no induction.
4. E.coli with ddpX gene, IPTG added as induction.
Then we continuously measure the OD₆₀₀ of the culture medium by 96-well Microplate Reader and draw the OD₆₀₀-culturing time curve by Matlab. The graph is showed below.

Fig.2. OD₆₀₀-culturing time curve of different groups of inclusion body induced cell lysis system.

3.Results of TPA positive feedback regulation system.

Summary