In order to produce our compartments and obtain a good yield, we opted for the E.coli strain BL21, which is a commonly used expression strain for recombinant proteins expression [78]. BL21 cells are a cell line of E.coli, usually multiply quicker than other cell types. In fact, BL21 cells lack flagella due to a significant loss in fli genes, which are necessary for the manufacture of flagellar proteins [79].
According to some scientists, this partly explains why BL21 cells grow faster, as the process of flagellar production and assembly requires a lot of energy. Furthermore, a second T2S secretion pathway allows BL21 cells to release more proteins than other experimental strains [80], that is why secretion of the foreign proteins shows sometimes better results than cytoplasmic synthesis [80][81].
In particular, we used the variant BL21(DE3), which lacks two important proteases, leading to the reduction of the degradation of heterologous proteins expressed in the strain. Plus, phage T7 RNA polymerase (T7RNAP), which is regulated by the lacUV5 promoter, is present in the BL21(DE3) strain [78][80], and is much faster than the E.coli RNA polymerase, which goes to our benefit [82].
When the free inducer isopropyl-D-1-thiogalactopyranoside (IPTG) is added to expression plasmids with genes of interest cloned under the control of a T7 promoter, protein synthesis can be initiated. This technology gives us complete control over the high selectivity and activity of protein synthesis induction. All of these characteristics made BL21(DE3) the ideal host [80].
For our project we also used the E.coli strain MG1655. By using this strain, we wanted to test if our toolbox could be formed in what is the most common laboratory K-12 strain, which approximates the E.coli wildtype, as sequenced by the Blattner laboratory [83].
This genomically recoded MG1655 strain was a gift from George Church (Addgene plasmid # 49018) [84]. This strain was designed to use for studies using non-standard amino acids. All 321 UAG stop codons (changed to UAA) and release factor 1 were removed from its genome (E. coli MG1655 Delta (ybhB-bioAB)::zeoR Delta prfA).
C321.ΔA.exp was a gift from George Church (Addgene plasmid # 49018).
To not overload the bacteria with the new proteins, we decided to test our toolbox on genome reduced bacteria. In this kind of organisms, genes that are not essential for growth are eliminated and energy and substrates that are usually required for less productive purposes are made available for engineering goals.
In fact, researchers found that E. coli bacteria can survive with approximately 30% of their genome removed. Genome-reduced bacteria have several benefits in biotechnology and synthetic biology, since more recombinant genes can be inserted into chromosomes and fewer cell resources are dedicated for uses other than biotechnological ones [85].
Additionally, the ability to enhance the genetic circuits may be controlled by removing unneeded genes and/or metabolic pathways [86][87].
For cells maintained in a stable environment in the lab, the genomic portions that have contributed to adaptation to external disturbance, such as stress response genes [86][88], insertion sequence (IS) elements and transposons, appear to be unneeded [89]. Due to adverse genome alteration, the ISs and transposons in particular may produce unexpected mutagenesis and be problematic in genetic engineering [90][91].
Additionally, it was hypothesized that the preservation of these superfluous genes would raise the cost of DNA replication [92][93] and that the expression of superfluous genes would hinder microbial development [94]. These unnecessary genomic regions might be removed, leaving cells with a clear genetic makeup and straightforward metabolic pathways [86].
Genome reduced bacteria can also be further metabolically engineered to improve the production of certain proteins or substances. In fact, for example, by removing the native threonine dehydrogenase and threonine transporter genes, as well as inserting a mutant threonine exporter gene, feedback-resistant biosynthetic enzymes were overexpressed in the genome reduced E. coli strain MDS42 in order to increase threonine synthesis. Surprisingly, l-threonine production by the resultant strain MDS-205 was 83% higher compared to the wild-type. This proves that by eliminating non-essential genes, it is possible to produce production strains that are suitable for the industrial world [95][96].
Kato et al (Tokyo Metropolitan University Group) constructed a series of large-scale chromosomal deletion mutants of E. coli (derivative of MG1655) that lack between 2.4. and 29.7% of the parental chromosome [97][98][99].
The following strains we used to express our microcompartments:
- ME 5000 (parental, non-deleted strain)
- ME 5010 (2.4% deleted)
- ME 5119 (15.8% deleted)
- ME 5125 (29.7% deleted)
During our research about genome reduced bacteria, we came across the Clean Genome MDS69 strain from Scarab Genomics. This strain has 20.32% of its genes deleted and is derived from the MDS42 genome reduced strain described in [100], which is a derivate of MG1655 as well.
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iGEM Freiburg 2022 presents
chAMBER
