Engineering Success

For the Engineering Success part, we focused on the alpha-amylase (AmyH) part: BBa_K4187022 composed of the AmyH coding sequence BBa_K4187016, the T7 promoter BBa_I719005, the strong RBS.1 BBa_B0030 and the double terminator BBa_B0015.

Cycle 1

Design:

First, we designed our sequence of the alpha-amylase used (AmyH) from the bacterium Halomonas meridiana on the SnapGene software (see Figure 1). We added the PstI and SacI restriction sites to the coding sequence ends to be able to extract and clone the sequence and further assembly. We want to overexpress an amylase in Escherichia coli to degrade the remaining starch in the brewers’ spent grain and to produce glucose.

Sequence of the initial composite part of AmyH with the T7 promoter

Figure 1: Sequence of the initial composite part of AmyH with the T7 promoter, the strong Ribosome Binding Site (RBS) and the double terminator made in SnapGene. The size of the sequence was 1634bp.

Build:

Once we received our sequence in a pUC19 plasmid from Integrated DNA Technologies (IDT), we transformed E. Coli DH5α to amplify it and bank it.

Test:

Then, to check our sequence of interest, we did a mini-prep, followed by a digestion using the restriction enzymes SacI and PstI, and ended by an electrophoresis of the digestion. As expected, we obtained two bands (see Figure 2). Once checked, we transformed E. Coli BL21 to try a functional characterisation by performing a Lugol test on Petri dishes with a non-transformed E. coli BL21 negative control and with a transformed E. coli BL21 with AmyH.

The AmyH digested with SacI and PstI

Figure 2: The AmyH digested with SacI and PstI. The DNA ladder used is the 1 kb DNA Ladder from Promega that is defined on the left. The expected size of the AmyH was 1,614 bp. The observed bands are above 1,500bp. These bands correspond to the expected size of the corrected AmyH.

Learn:

During our functional characterisation, we found no significant effect from AmyH compared to our control. Thus, we decided to check our designed sequence. We found that there was an issue with our T7 promoter: it was truncated (see Figure 3). The amylase could not be expressed. In fact, our truncated T7 promoter size was 10bp instead of 23bp.

Comparison of our truncated T7 promoter with the T7 promoter

Figure 3: Comparison of our truncated T7 promoter with the T7 promoter made on Benchling

Cycle 2

Design:

To fix our promoter, we designed and ordered primers to be able to fix the issue on the alpha-amylase with the truncated T7 promoter via Polymerase Chain Reaction (PCR) (see Figure 4).

Sequences of our Forward and Reverse primers to correct T7 promoter of AmyH

Figure 4: Sequences of our Forward and Reverse primers to correct T7 promoter of AmyH done on SnapGene

The fixation of both primers, forward and reverse, on the AmyH composite part with the truncated T7 promoter 1 The fixation of both primers, forward and reverse, on the AmyH composite part with the truncated T7 promoter 2
The fixation of both primers, forward and reverse, on the AmyH composite part with the truncated T7 promoter 3

Figure 5: The fixation of both primers, forward and reverse, on the AmyH composite part with the truncated T7 promoter from SnapGene. We deleted the parts that we had originally planned for the Golden Gate assembly, so we expect to see a band at 1629pb.

Build:

We performed a PCR on the alpha-amylase with the truncated T7 promoter, and analyzed it by electrophoresis. We used the protocols: Polymerase Chain Reaction using Q5® High-Fidelity 2X Master Mix of NEB and Electrophoresis (Gel preparation and migration) in the Experiments part.

Test:

The electrophoresis gel showed the expected bands of AmyH, 1,629 bp, (see Figure 6), so we tried once again to assemble it in the pmScarlet-i_C1 (Plasmid #85044 on Addgene) [2] plasmid (see Figure 7 and 8) to try our functional characterisation once again.

Gel electrophoresis of the corrected amplified AmyH and LDH

Figure 6: Gel electrophoresis of the corrected amplified AmyH and LDH done on 09/27/2022. The DNA ladder used is the 1 kb Plus DNA Ladder from NEB that is defined on the left. Both T7 promoters of LDH and AmyH were corrected via PCR. Without counting the last well, the first six wells correspond to AmyH. The expected size of the AmyH was 1,629 bp. The observed bands are slightly above 1,500bp. These bands correspond to the expected size of the corrected AmyH. In the fifth well of AmyH, the PCR did not work probably because of the absence of DNA.

pmScarlet-i_C1 plasmid used as a vector for the assembly of AmyH

Figure 7: pmScarlet-i_C1 plasmid used as a vector for the assembly of AmyH. This backbone has the two restriction sites SacI and PstI to assemble correctly AmyH digested with those restriction enzymes

Picture of the Petri dish with the ligation of digested AmyH and digested

Figure 8: Picture of the Petri dish with the ligation of digested AmyH and digested pmScarlet-i_C1 plasmid. The colonies are pink because the Red Fluorescent Protein (RFP) is still expressed in our ligation.

Learn:

This time, the characterisation yielded good results (see Figure 9) (see Proof of concept for more). Therefore, we attempted to assemble our whole Depolymerization plasmid (see Figure 7, Parts collection).

Pictures of our characterization of AmyH

Figure 9: Pictures of our characterization of AmyH under the same conditions with E.Coli BL21 transformed with pmScarlet-i_C1 plasmid containing the AmyH composite part and untransformed E.Coli BL21 (wt). Salt was added because the amylase that we chose has an optimal activity with salt [1]. We dyed the starch with a lugol solution. Starch concentration: 0.2 g/L NaCl concentration: 5% (w/v) NT BL21 mean halo surface = 0.05 cm²; amyH BL21 mean halo surface = 1.25 cm²

Cycle 3

Design:

We had decided to assemble the Depolymerization plasmid in the pSB1A3 plasmid, and had chosen the appropriate enzymes to perform the Digestion-Ligation assembly.

Build:

In our Depolymerization plasmid, we had three inserts and one plasmid to digest. AmyH was once again digested with SacI and Pst1.

Test:

We tested our digestion by electrophoresis to check our ligation. We obtained the appropriate bands on our electrophoresis, but when we purified our gel, we obtained weak concentration (>5 ng/uL). We still tried to perform our ligation, but it wasn't successful as nothing grew on the Petri Dish (see Figure 10).

Picture of a Petri dish containing E.Coli BL21 with the Depolymerisation plasmid

Figure 10: Picture of a Petri dish containing E.Coli BL21 with the Depolymerisation plasmid. No colony is observed. The ligation did not work.

Learn:

We learned that we have to try again the ligation by increasing the concentration of the vector and the inserts. To do that, we will try other ratios as 1 vector:1 insert, 1 vector: 2 inserts for the ligation. We will do other mini preps of pSB1A3, EL222, PLAase, AmyH to have a higher quantity of plasmid and inserts. If it does not work, we should order primers to amplify our purified fragments,the plasmid and the inserts, to have a higher concentration.

These cycles permit the correction of a few issues. However, our last engineering success cycle was not successful, other cycles will probably be needed to succeed the depolymerization plasmid assembly.

References

  1. María-José Coronado, Carmen Vargas, Jürgen Hofemeister, Antonio Ventosa, Joaquín J. Nieto, Production and biochemical characterization of an α-amylase from the moderate halophile Halomonas meridiana, FEMS Microbiology Letters, Volume 183, Issue 1, February 2000, Pages 67–71.
  2. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M, Aumonier S, Gotthard G, Royant A, Hink MA, Gadella TW Jr. Nat Methods. 2016 Nov 21