Part:BBa_K4034009
pAdapted, a plasmid for the production of dNTP's
no
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 296
Illegal EcoRI site found at 5147
Illegal EcoRI site found at 5747
Illegal PstI site found at 234 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 296
Illegal EcoRI site found at 5147
Illegal EcoRI site found at 5747
Illegal PstI site found at 234
Illegal NotI site found at 5158 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 296
Illegal EcoRI site found at 5147
Illegal EcoRI site found at 5747
Illegal BglII site found at 2287
Illegal BamHI site found at 290
Illegal BamHI site found at 4097
Illegal XhoI site found at 284
Illegal XhoI site found at 5152 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 296
Illegal EcoRI site found at 5147
Illegal EcoRI site found at 5747
Illegal PstI site found at 234 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 296
Illegal EcoRI site found at 5147
Illegal EcoRI site found at 5747
Illegal PstI site found at 234
Illegal NgoMIV site found at 704
Illegal AgeI site found at 891
Illegal AgeI site found at 1233
Illegal AgeI site found at 1632
Illegal AgeI site found at 3453
Illegal AgeI site found at 4233
Illegal AgeI site found at 4239
Illegal AgeI site found at 4773 - 1000COMPATIBLE WITH RFC[1000]
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.
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.
RNR
Function
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.
Isolation and Purification
Since Pfu is an important enzyme worldwide there have been reported many attempts to produce it and purify it. Originally Pfu polymerase was isolated directly from Pyrococcus furiosus, but growing this species is a challenge especially in large quantities [9]. Thus, it has been successfully attempted to express that enzyme in E. coli BL21 [10]. The purification of the protein has been done with many different ways like His-tag purification and with the use of weak cation exchange resins [11, 12]. Recently a new simple method utilizing the tolerance to heat of the enzyme has been used successfully introducing a new level of simplicity for isolation and purification of thermotolerant proteins [13].
Histidine Tag
In the Pfu encoding gene, a histidine tag is added to isolate the protein for future applications. Purification of Pfu using polyhistidine affinity tags was selected as it is a rapid and efficient method, resulting in 100-fold enrichment and up to 95% purities in a single purification step (Bornhorst et. al. 2000).
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