UMaryland iGEM submitted various basic and composite parts to the BioBrick Registry, which aims to increase standardization in synthetic biology by allowing genes to be added together easily. We synthesized the genes, put them inside standard BioBrick plasmids, and then characterized our parts.
Basic Parts Composite Parts
This composite biobrick part is a combination of the coding regions for three separate enzymes involved in the metabolization of methanol. Each coding region is preempted by a ribosome binding site in order to help counteract some of the translational issues associated with polycistronic mRNA. A lacI regulated promoter that allows for induction with IPTG was included in this construct to allow for selective gene expression along with the standard iGEM double terminator.The three enzymes encoded by this part are methanol dehydrogenase 2 (MDH), 3-hexulose-6-phosphate synthase (HPS), and 6-Phospho-3-hexuloisomerase (PHI). These enzymes serve to function as a three step pathway in which methanol is metabolized. MDH converts methanol to formaldehyde, producing a molecule of NADH in the process. Formaldehyde is then combined with a molecule of D-ribulose-5-phosphate taken from the pentose phosphate pathway to form one molecule of D-arabino-3-hexulose-6-phosphate via the usage of HPS. PHI then converts D-arabino-3-hexulose-6-phosphate to D-fructose-6-phosphate which can then undergo glycolysis.
The Formate Pathway is a three enzyme pathway that begins with methanol and NAD+ as substrates, and culminates in the production of NADH molecules and carbon dioxide. This pathway is used to detoxify alcohols in the cellular environment. The pathway consists of a series of oxidations: methanol oxidized to formaldehyde by Methanol Dehydrogenase 2 (MDH2); formaldehyde oxidized to formate by Formaldehyde Dehydrogenase (FALDH); and finally formate oxidized to carbon dioxide by Formate Dehydrogenase (FDH). Each of these catalyzed reactions results in lower energy products than reactants, so every reaction is coupled to the production of one NADH molecule, which contributes to energy for the cell and biomass production. The first enzyme of the pathway, MDH2, has a low binding specificity and will oxidize many primary alcohols. The original use of this plasmid was to detoxify methanol as part of a larger pathway that consisted of eliminating methane gas from the atmosphere. The pathway has potential uses for detoxifying alcohols in the environment as well. As methanol becomes an increasingly popular liquid fuel source, one could imagine methanol spills in the future that require detoxification. This part contains one promoter that is IPTG inducible. The genes will be transcribed as a polycistronic mRNA strand. Each of the genes has a medium strength ribosome binding site. Modeling of this pathway has revealed that no toxic substrates should be produced when this pathway is expressed. The kinetics for each enzyme exist in such a way that there will be no buildup of formaldehyde of formic acid in the cell. Theoretically, this pathway should increase methanol resistance to cells that express it.
This is an intermediate used in the construction of BBa_K2032002. It contains the coding sequence for MDH2 which oxidizes methane to formaldehyde. It also contains the lac + pL promoter (BBa_R0011) which is repressed by LacI and induced by IPTG.
Fructose construct with RFP reporter
This biobrick contains the coding regions for the Y and Z subunits of the sMMO enzyme. Each coding region is preceded by a ribosome binding site (BBa_B0032) the whole biobrick is flanked by the standard iGEM double terminator (BBa_B0015 )