Results

At first sight, we decided to do the Golden Gate assembly but we decided to choose a simple method. For the assembly of our BioBricks, we did a method that is widely used: the BioBrick Assembly (Restriction Enzymes) method. For that, we designed our different coding sequences with the basic parts (Ribosome Binding Site (RBS), a T7 promoter, a double terminator) and with the restriction enzyme sites we wanted.

a. Polymerization Plasmid

For the polymerization plasmid (http://parts.igem.org/Part:BBa_K4187029), corresponding to pSB1K3) , our BioBrick is composed of the following composite parts: a D-Lactate Dehydrogenase (LDH) (http://parts.igem.org/Part:BBa_K4187017), followed by a propionate-CoA transferase (Pct) (http://parts.igem.org/Part:BBa_K4187018) and a Polyhydroxyalkanoate (PHA) synthase (PHAC1) (http://parts.igem.org/Part:BBa_K4187019).

Our first step was the digestion of each of our gene fragments with the corresponding restriction enzymes. LDH was digested with XbaI and BamHI. Pct was digested with BamHI and SacI. PHAC1 was digested with SacI and PstI. The pSB1K3 was digested with Xba1 and Pst1 (see Figure 1).

For LDH, we encountered two problems in our sequences: there was a methylation on XbaI restriction site and the T7 promoter did not have all of its sequence. To correct it, we ordered primers and we did a Polymerase Chain Polymerase (PCR).

Once corrected, we have to digest the amplified fragment of LDH with the restriction enzymes: XbaI and BamHI. However, we did not have any DNA ladder that could permit us to check the digestion of LDH in our stock.

Because of the impossibility of checking the digestion of the corrected LDH, we directly tried the assembly of our polymerization BioBrick.

b. Depolymerization plasmid

For the depolymerization plasmid (http://parts.igem.org/Part:BBa_K4187024) corresponding to pSB1A3, our BioBrick is composed of Light-activated DNA-binding protein (EL222) (http://parts.igem.org/Part:BBa_K4187020), followed by the Blue light inducible promoter (Pblind), an inducible promoter, that is activated by EL222 inducing the expression of a Polylactate depolymerase (PLAase) (http://parts.igem.org/Part:BBa_K4187021), and then completed with an alpha-amylase (AmyH) (http://parts.igem.org/Part:BBa_K4187022).

The purpose was also to make this construction by also using the restriction enzyme method. EL222 was digested with XbaI and NcoI. PLAase was digested with NcoI and SacI. AmyH was digested with SacI and PstI.

For these results on EL222, we checked the sequence and we saw a methylation on the XbaI site preventing the cut by XbaI. We ordered the correct sequence without the methylation. However, we received EL222 as a gene fragment. So we could not check if it was correctly digested or not.

As LDH, we could not check the digestion, so we tried directly with the purified band of EL222 obtained for the assembly.

For AmyH, we encountered the same problem with the T7 promoter, so we had to correct it via PCR. We designed the primers for it.

Then, we digested the amplified AmyH. But, we encountered the same problem as LDH. We could not check that our amplified AmyH is digested.

Once AmyH is digested, we tried the assembly of the depolymerization plasmid as well as the polymerization plasmid.

However, we did not succeed to do our assemblies.

We assessed the ability of E.Coli BL21 transformed with the amyH (amyH BL21) gene to degrade starch by performing a lugol staining assay on solid cultures. As this amylase’s activity depends on NaCl concentrations, we tested multiple salt concentrations (i.e. 0%, 5%). We cultured the bacteria as well-defined separated colonies on solid media containing starch. We defined degradation halos as the total unstained surface minus the surface of the colony: S(unstained) - S(colony).

We measured these surfaces using ImageJ, an open source software developed by Wayne Rasband.

Our results show an increased starch degradation activity in E.coli BL21 transformed with AmyH as the mean halo sizes of the colonies are higher in transformed colonies.
We cannot conclude on the NaCl concentration effect on the amylase activity as the maximum mean halo sizes are similar in both NaCl concentration conditions. However we observe that with a 5% NaCl concentration, halo sizes of non-transformed BL21 are close to zero while halo sizes of transformed BL21 reach the same value as, in the 0% NaCl condition.

We tested the ability of Shewanella oneidensis to generate an electric current in varying conditions. We tested the effect of lactate, riboflavin, and the mtrC gene overexpression against a non-transformed Shewanella oneidensis without any lactate in the medium. We performed these measurements using our first prototype described in Figure 15. As our hardware was not sensitive enough to measure intensity in our working ranges, we decided to measure voltage and made the assumption that our system’s resistance remained the same throughout the experiment.

All conditions plateaued at their maximum tension variation 20 to 30 minutes after the beginning of the experience. The maximum tension variation of 28mV was observed with Shewanella oneidensis transformed with the mtrC gene and supplemented with 37nM riboflavin. This shows us that riboflavin might be the best way for improving electron transfer as it reaches a tension of over 10 mV above all the other conditions.

No significant difference was observed between Shewanella oneidensis transformed with mtrC and non-transformed Shewanella oneidensis which both plateau at around 16mV.
Shewanella oneidensis transformed with p150 plateaued at around 5mV. Non lactate-supplemented wild type shewanella oneidensis did not show any consistent tension plateau and showed a max tension of 3mV. After 9 hours, all MFC returned to their baseline tension.

The results from the co-culture of E.coli secreting Phenazine-1-carboxylic acid and Shewanella oneidensis were not what we were expecting, especially compared to Shewanella oneidensis with Riboflavin. Indeed, we expected to see better results coming from the Phenazine-1-carboxylic acid. However, the test with Phenazine-1-carboxylic acid involved a co-culture of Shewanella oneidensis and E. coli, and we suspect that this is why we obtained weak results. We believe that E. coli may have overgrew Shewanella oneidensis.

First, we performed a cell lysis to extract the proteins. Then, to obtain the concentrations of our samples, we carried out a standard curve by using Bradford reagent measuring the absorbance of known concentrations of BSA. (see Figure 17)

Once we got this curve, we were able to get the concentration of our samples by measuring their absorbance. Depending on their concentration, the samples were diluted by 2 or by 5. Then, in order to get optimal results in our SDS-Page, we wanted 20 ug of proteins per well. The results can be seen in Figure 18.

After checking the amount of proteins in our samples, the R&D team thought about testing our proteins using an SDS-Page technique in order to isolate and see our protein of interest.

Many SDS-Pages were performed in order to characterize our 7 sequences one by one. But first bibliographical research are needed to find the molecular weight of our part sequences:

• PHAC1: between 48 and 63 kDa [1]
• AmyH: 50.189 kDa [2]
• PCT: 67 kDa [3]
• EL222: 24.741 kDa [4]
• LDH: 37.049 kDa [5]
• PlAase (RPA1511): 34, 1 kDa [6]
• mtrC: 71.237 [7]

As you can see on the Figure 19, the first control of E.Coli BL21 wt in position 1 worked well since no strip is bigger than the others in the first well. We will use this well as a reference (negative control) to compare to others Electrophoresis Gel.

When we are comparing the Figure 19 with the Figure 20, we can admit that the 4th well is corresponding to the part named EL222 since one strip is thicker than the others and her molecular weight is around 24 kDa.

Now we can observe that on all the wells corresponding to PCT (from number 8 to 10), a thicker strip just below the strip of the molecular weight corresponds to 70 kDA (Figure 20).

Even if our electrophoresis gel is kind of blurry with the first test (Figure 19) we can assess that BL21 wt worked well as a negative control. And looking at the characterization of our part; EL222 is expressed in our construction as well as PCT since the proteins of interest produced are over-expressed in the electrophoresis as we can see on the Figure 19 at the corresponding Molecular Weight.

As we can observe on this second gel the BL21 wt, our negative control worked well since no strips are overexpressed in the 7th well.

When we compare the 7th well and the molecular weight in the 6th position a thicker strip is seen in the 4th well corresponding to the loading of the transformed bacteria with PHAC1. In fact the thicker band is between the molecular weight of 55 kDa and 70 kDa (Figure 20), and corresponds to the expected molecular weight of PHAC1 which is between 48 kDa and 63 kDa.

Moreover, on the 5th well corresponding to AMYH we can observe a thicker strip between the molecular weight (6) of 35 kDa and 55 kDa (Figure 20), that is around 50kDa which correspond to the molecular weight of the expressed protein AMYH leading to the validation of our engineering success.

On the fifth well, we observed a band slightly above 55 kDa that could correspond to LDH because its molecular weight of the expressed LDH is at 57 kDa. (see Figure 20) We know that E.Coli expresses its own Lactate dehydrogenase which has a Molecular weight around 36kDa [8]. On the first gel, we observe the same bands as here that explains why. We can suppose that our LDH is expressed with the support of its characterisation in the Proof of Concept part

As a conclusion on the SDS Page performed by the team, we succeed in proving the expression of most of our parts such as AMYH, EL222, PHAC1, LDH and PCT. This may validate the right construction of our sequences. But other sequences such as PLAase and mTRC still need to be characterized on another electrophoresis gel.

Since mTRC was transformed into Shewanella oneidensis, the wet lab team should have loaded a “Shewanella oneidensis wt” as a negative control in the electrophoresis gel to properly interpret the mTRC expression or not, but due to a lack of time we didn’t perform it yet.