Revision as of 09:44, 17 October 2021

pAdapted, a plasmid for the production of dNTP's

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Sequence and Features

Description

This part was constructed for the project AdAPTED of iGEM Athens 2021. Its goal is to result in overexpression of RNR and TSase enzymes, when E. coli BL21 bacteria are transformed.

pGGA

This backbone contains a Chloramphenicol resistance gene under the control of a cat promoter. Additionally it contains a high-copy number ori and a SP6 promoter and a T7 promoter flanking two bsaI cut sites. Lastly, it contains two MCS between outside the two bsaI cut sites.

RNR

RNR catalyzes the conversion of all four ribonucleotides triphosphates (NTPs) into the corresponding dNTPs, therefore providing the building blocks for the synthesis and repair of DNA. This conversion is achieved by the reduction of the C2’-OH bond. This biochemical pathway is the only de novo dNTP production method. The reduction occurs for all nucleotides at a single active site. RNR is divided into three classes I, which is further divided into class Ia, Ib, and Ic, II, and III.

Protein Structure

Class I RNR is composed of two homoderic subunits α and β. When active, both in eucaryotes and procaryotes, the two proteins are associated in a dimeric or other oligomeric form, such as (alpha)n(beta)m. Based on the subclass of RNR, the metal centre required for the radical production, differs. Despite the differences apparent in the classes of RNR, all three contain a conserved cysteine residue at the active site. This cysteine residue is possibly converted into a thiyl radical, initiating the substrate turnover, by abstraction of a hydrogen atom from a ribose ring of the substrate. The substrate binding active site is located in the alpha 2 homodimer, encoded by nrdA. The binding site for the two irons is contained in the beta 2 homodimer, encoded by nrdB.

Ia RNR

RNR Ia is dependent from oxygen, contains a di-iron center (FeIII-O-FeIII), has two allosteric centers, can be inhibited by ATP, is distributed in Eukaryotes, eubacteria, archaea, bacteriophages, and virus, is either in the (alpha)2(beta)2 or (alpha)6(beta)6 form, and is encoded by the nrdA and nrdB genes.

nrdA

The alpha subunit contains the catalytic subunit, with the active site, for the nucleotide reduction. It also contains two allosteric sites for the allosteric regulation of RNR.

nrdB

The beta subunit contains the metallocofactor (di-iron), required for the reduction initiation.

TSase

Thymidylate synthase (TS), has the ability to bind with cellular RNA, forming a complex, which results in translational repression. In general, TS plays an important role in the regulation of the cell cycle, translation, chemosensitivity and apoptosis. Lastly, TS catalyzes the reduction of deoxyuridylate (dUMP) to thymidylate (dTMP), the only de novo method of dTMP production.

Protein Structure

A three secondary stem - loop structure is observed, and this exact structure is hypothesized to influence the TS mRNA translation. With deletion of any one of these repeated sequences, the translational TS mRNA efficiency was altered. The secondary structure of TSase is also important for protein recognition.

T7-LacO

It was important for the expression of both of those enzymes to be regulated. Even though we want to overexpress RNR the increased concentration of dNTP’s can prove fatal for the bacteria due to mutations. Thus we need to control the expression of these genes. For this purpose the T7-LacO promoter is chosen with whom we can control the expression of the genes regulating the concentration of IPTG in the medium. The part that was utilized was BBa_K2406020.

AraC

AraC is used to regulate TSase so an equal level of dNTP's production is achieved. More about this part on BBa_R0080.

RBS

The RBS (BBa_B0030) was chosen from the existing parts of the iGEM Registry, due to its high popularity and efficiency for E. coli.

Terminator

The part that was used was taken from the iGEM Registry BBa_B0015.