Engineering cycles
Design
PET accounts for 12% or all solid waste materials worldwide (George, N. & Kurian, T. 2014) even though it could be theoretically integrated into a recycling or upcycling process. Since enzymatic depolymerisation possess great potential for the utilization of PET waste it was decided to attempt the integration of all enzymes needed for PET upcycling into PCA as an easy to handle and cheap option. After extensive research our team identified the necessary enzymes for this task and thought of a design to assemble those enzymes in a single organism as it has never been done before. To allow characterization of all the necessary enzymes individually in sufficient amounts all were designed to have the cassette necessary for inducible protein overexpression through the T7-RNA-Polymerase system. All the coding sequences (CDS) were carefully collected and codon-optimised for expression in E. coli and contained the following elements: BioBrick prefix, containing restriction sites for EcoRI and XbaI, and suffix with sites for restriction by SpeI and PstI were added on ends of the construct for easier insertion into vectors. Our construct was also designed with RBS, T7 promoter and terminator and lac operator for optimal induction of protein expression(Du, L., Villarreal, S. & Forster, A. C. 2012) (Shepherd, T. R. et al. 2017) and C-terminal histidine tag for enzyme purification on immobilized metal affinity chromatography.
Problem
Initially the CDS of PETase, MHETase and LCC were ordered from Integrated DNA Technologies (IDT) and designed to be compatible with any BioBrick vector. After the order was shipped, it was noticed that the necessary flanking regions next to the restriction sites to allow effective restriction were missing.
Fix
Fortunately, this mistake was noticed early enough to design primers which could introduce extra base pairs in both ends of the construct by annealing to the prefix (fwd) or suffix (rev). Since all the constructs initially ordered ended with the prefix and suffix one universal primer pair was able to fix the constructs via PCR. PCR primers used were:
Forward 5’-NNNNNGGAATTCGCGGCCGCTTCTAG,
Reverse 5’-NNNNNNCTGCAGCGGCCGCTACTA-3’.
Further orders from IDT were made containing the necessary flaking regions.
Design
To allow proper overexpression of the different enzymes the now fixed sequences should be ligated into a pET24a vector which was modified in 2009 to be compatible with BioBrick cloning.
Problem
The transformation plates usually showed no or very few colonies after one night of incubation. The few colonies which grew also showed in restriction analysis religation of the pET24 vector instead of the desired assembled construct.
Fix
To overcome this problem NEB® 5-alpha Competent E. coli (High Efficiency) were used. Those showed after transformation high amounts of colonies. Screening of those colonies showed that usually 25-50% of those contained the correct assembled construct. This showed that the cloning protocols used showed low efficiency which could be caused by enzymes with lowered activity due to suboptimal storage and handling. Another factor which could have had influence on our results was the before mentioned PCR to fix the construct as the PCR samples needed gel purification due to undesired side products. These purification usually resulted in low yield and impure DNA samples. The commercial cells which had a transformation efficiency 10^4 higher than our prepared cells most likely just overcame the low efficiency of the cloning procedure by its efficiency.
Design
As multiple Lab groups struggled with the use of pET24a as a vector they changed to a pSB1C3 vector containing a cassette for constitutive protein expression of the chromoprotein mRFP1 as it allowed easier screening for successful transformation by color of the colony instead of restriction analysis.
Problem
After a Lab group succeeded cloning the fixed construct into pSB1C3 they attempted protein overexpression. Multiple tries didn’t show any sign of expression of the desired protein in SDS-PAGE analysis even though an overexpression of the T7-Polymerase in the BL21 cells was observed. The reason for this wasn’t ultimately found.
Fix
The same expression cassette was later cloned into a pET24a vector. With this vector which is specifically designed for overexpression a band at the desired size was observed in SDS-PAGE which shows the importance of expression vectors over vectors designed for different purposes.
Our team went through the mentioned engineering cycles over the course of our lab period to overcome these obstacles. In the end these efforts paid of as we managed the overexpression of two of our enzymes and one of them showed a surprisingly high over expression. Many of these problems we encountered were new to us, but were overcame them through troubleshooting. In some cases when we were unsure about the root cause of the problem but controls allowed us to come up with reasonable hypotheses. Through using the scientific method and eliminating possibilities, we managed to make progress towards our set goals. Overall we learned a lot during this summer and especially through these engineering cycles.
Du, L., Villarreal, S. & Forster, A. C. Multigene expression in vivo: Supremacy of large versus small terminators for T7 RNA polymerase. Biotechnology and Bioengineering 109, 1043–1050 (2012).
George, N. & Kurian, T. Recent developments in the chemical recycling of postconsumer poly(ethylene terephthalate) waste. Ind. Eng. Chem. Res. 53, 14185–14198 (2014).
Shepherd, T. R. et al. De novo design and synthesis of a 30-cistron translation-factor module. Nucleic Acids Research 45, 10895–10905 (2017).
