Engineering Success

Intro

Every engineering process consists of multiple milestones that together build the final system. Engineering a biological system is not straightforward and often requires many customizations and fine tunings to achieve optimization. In Fig.1 you can see the engineering cycle that describes the main stages in every engineering process. In our case, our end system is an E. coli BL21 strain which has two plasmids that express three enzymes: Umbelliferone-6-prenyl-transferase, XimD, and XimE. Read more in our Design page.

U6PT plasmid

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In order to build our entire biological system, we worked on both plasmids in parallel. Design, build, test, learn and re-design. This was our strategy to tackle all the wet lab obstacles. Starting with the challenging cloning of the first enzyme Umbelliferone-6-prenyl-transferase (U6PT):

	1st Design

U6PT genes are plant sourced. Thus, in order to enlarge our chances of success, six different U6PT genes were used; four of which originate from different plant organisms and two were constructed by us using BLAST and docking computational tool, for more details, read in more in our Design page.

All were designed to be cloned into a plasmid that has mCherry reporter gene downstream to the P2A sequence (fig.2-fig7).

	1st Build

Step1: cloning the P2A sequence upstream to the mCherry sequence into A133 plasmid.
Step2: cloning All U6PTs genes upstream to the P2A sequence.

	1stTest

Each cloning was verified using colony PCR. The results have shown positive insertion for the P2A sequence (fig.8-Colony PCR gel for the verification of P2A cloning) but negative results for all the U6PTs genes (fig.9-Colony PCR gel for the verification of All U6PT cloning).

	1st Learn

The ligation was not successful.

	2nd Re-design

We decided to tweak the ligation protocol, using different ligation ratios between the insert and the backbone (1:7 rather than 1:3).

	2nd Re-Test

Another negative Colony PCR results- Self ligation products

	2nd Re-Learn

The P2A sequence might hinder the ligation of the U6PT genes.

	3rd Rre-design

Ligation of three fragments: The digested backbone (A133), one of U6PT genes and the P2A sequence. Thus, the two inserts will be cloned simultaneously.

	3rd Re-Test

Colony PCR results have shown self-ligation products only. A digestion test also was conducted to make sure that all the required restriction sites are found in the backbone. Indeed, all of them were found but they are very close (402bp).

	3rd Re-Learn

We have learned that in order to get the desired restricted product. A two satge of digestion should be done in order to avoid a steric hindrance. After sending the colony PCR products for sequencing, it was shown that the cloning of U6PT genes interrupts the sequencing process. Meaning, Sanger sequencing failed after less than 400 bases (fig.10-sequencing results).

To further investigate this situation, we set aside five of the six U6PTs, and continued with PcPT, for the purpose of pinpointing the problem more efficiently.

PcPT was the only U6PT out of the six that was not ordered as a DNA fragment, rather it was amplified from a plasmid using PCR.

	4th Re-design

We have re-designed the new genetic U6PT circuit in a way that will allow us to clone the PcPT gene into the same target backbone but after digestion of the mCherry sequence out of the plasmid(fig.11-PcPT genetic circuit)

Step1: Amplifying the PcPT gene with new restriction sites.
Step2: A133 backbone digestion with the new restriction enzymes.
Step3: Ligation of two fragments.
Step4: Transformation

	4th Re-Test

As mentioned previously ,every cloning of a specific fragment was verified using Colony-PCR. However,we have gotten undesierd prodcuts.

Unfurtionatly,Many steps were not accomplished, and many others were skipped. But we are aware of the fact that the process of engineering a biomanufacturing machine like ours is full of obstacles that we did not have enough time to tackle. However, we've created additional designs and future plans to continue with. To read more about our future plans click here

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ximD_ximE plasmid:

As mentioned in the Design page, the second reaction in our synthetic pathway is mediated by two enzymes, ximD and ximE. Both enzymes work together to convert 7-demethylsuberosin (DMS) into decursinol.

	1st Design

The two genes of interest: ximD and ximE were designed to be cloned into the same plasmid under the regulations of Lac and Tet respectively as shown in fig.12.

	1st Build

We started with pET_Duet_PcPT_ximD_ximE plasmid. The building of this circuit was done as shown below:
Step1: Reverse PCR to amplify the ximE gene out from the plasmid. This step was done in order to replace the gene of ximE with the same gene but under inducible regulation. Step2: Reverse PCR with primeSTAR GXL polymerase to amplify the PcPT out from the plasmid. This step was done multiple times using Q5 polymerase, but no successful amplifications were achieved. To solve this problem, we looked for a polymerase with the ability to amplify long amplicons. Eventually, primeSTAR GXL polymerase was the solution. It is the most high-fidelity polymerase that is used for challenging targets like: high GC content, long amplicons and excess template^[1].
Step3: Ligation of 3 fragments - pET_Duet backbone, ximE, T7 Terminator.

	1st Test

Tests were ran using ODE and statistical models based on Michales Menten equations as described on the Modeling page.

Modeling results have shown positive correlation between the concentration of decursinol (the desired product) and the concentration of XimE enzyme (fig.13).

	1st Learn

XimE should be expressed constitutively and not under any inducible regulation.

	2nd Re-design

To fulfill a constitutive expression for XimE enzyme, TetR repressor was not cloned into ximD_ximE plasmid as shown in fig.13.

To sum up, according to the final design for the second plasmid, ximD is expressed inducibiliy under the Lac operator, however ximE is expressed constitutively to achieve maximal biomanufacturing yield of decursinol. This in accordance to our model that predicts higher decursin to marmesin ratio when the molar fraction of XimE increases.

	2nd Re-Test

Real time qPCR assessment was run, following a transformation step into E. coli BL21 DE3. The purpose of this test is to determine the mRNA levels of each enzyme. XimE enzyme is constitutively expressed However, ximD expression is controled under the Lac regulation. So we added an IPTG molecule to induce the enzyme's expression. The qPCR results are presented in fig.16.

In the scope of iGEM, it was clear to us that achieving all of our milestones is a challenge due to the lack of time. Functionality tests and other modelling results were planed to be implemented in the designs of our genetic circuit. In our future plants page, all of our next steps and future proposed improvmments are presented.