Team:UCL/Project/Xenobiology

Designing Xeno-Coli

1. Choosing a metabolic pathway

In order to create a xenobiological organism with a metabolic firewall we decided to try to design cofactor that would be essential to E-Coli metabolism that could be derived from our Azo Dye waste products. This cofactor would need to be functionally similar to an existing molecule in the E-Coli metabolism.

Given the vast number of cofactors in the e-coli we needed a method to select the molecular structures closest to azo dye waste products. To solve this problem we developed a computer program that would do just that

2. Designing the Xeno-quinones

The pathway chosen from XenoRank was that of the cofactors ubiquinone and menaquinone. They are the electron buffer substituent of coenzyme Q in complex III of the electron transport chain that creates ATP on the membrane of E-Coli.

Ubiquinone is the electron buffer used in aerobic conditions and menaquinone in anaerobic conditions. Both quinols produce protons and electrons to create the electrochemical gradient used later in Complex IV and V to yield ATP.

In order to design Xeno versions of these molecules, it was important to ensure that we understood what each constituent on the the molecule provided to the chemical reaction.

From this understanding we designed the following molecules for our xeno-coli that we believe retains the properties described above

Once we The next step was to generate the organic chemical mechanism to go from the azo dye waste products to the new quinols. They had to be generated via organic chemistry as opposed to synthetic biology because if they could be produced by biological mechanisms they would not work as xeno molecules (please see science of xeno section).

3. Replacing the Natural Quinones

In order for our organism to become an autotroph to our xenobiological quinones it was necessary for us to remove the original quinones. The best approach it seemed was to do a gene knock out for a biosynthesis step of ubiquinone and menaquinone. The metabolic pathway provided a number of options

A search of the literature showed that a knockout of ispB successfully killed e-coli colonies. ispB codes for the protein that attaches the hydrophobic side chain on the quinones, allowing them to dock in the membrane. As ispB is used to synthesise all electron buffer quinones, it was the perfect knock out as it meant that only one was required.

Antisense RNA gene Silencing

Our first step was to try a proof of concept knockdown technique; Antisense RNA gene silencing. This is where the reverse complement of the coding strand is transcribed and interferes with the ispB mRNA hence affecting translation.

The design for the primers that would amplify the gene contained the bbk prefix and suffix. The forward primer contains suffix, reverse primer contains prefix hence sequence inserted as reverse complement into the vector

The gene was then cloned into pSB1C3 under the control of lac inducible promoter to observe effect on growth.

The knockdown showed to be unsuccessful in reducing bacteria growth and therefore cannot be use to create a xenobiological organism. The next step to try was a full knock out using the crispr technique

CRISPR Knock Outs

The CRISPR technique is based on the bacterial ability to cut out or replace viral DNA that has been inserted into it’s plasmids. We now can now utilise the Cas9 protein with a target sequence to do cut out of any gene within a plasmid.

The Cas9 protein is expressed with gRNA that is complimentary for the target sequence within the DNA you wish to cut out with a PAM (Protospacer Adjacent Motif) sequence downstream of the target. The PAM sequence in e-coli must be an NGG sequence (N representing any base)

To design our gRNA we used the target sequence online design tool DNA 2.0: https://www.dna20.com/eCommerce/cas9/input

Our gRNA target sequences were as follows:

Despite our plans there was not sufficient time to complete a knockout using CRISPR and that will be our next step beyond the competition.