Difference between revisions of "Part:BBa K4034009"
 
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=Description=
 
=Description=
 
<br>
 
<br>
−
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. <br>
+
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. The mRFP1 was added to measure the BBa_E1010.<br>
  
 
==pGGA==
 
==pGGA==
 
<br>
 
<br>
 
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. <br>
 
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. <br>
 +
 +
==RNR==
 +
 +
<br>
 +
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.
 +
<br>
 +
 +
===Protein Structure===
 +
<br>
 +
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.
 +
 +
<br>
 +
 +
===Ia RNR===
 +
<br>
 +
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.
 +
 +
<br>
 +
 +
===nrdA===
 +
<br>
 +
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.
 +
<br>
 +
===nrdB===
 +
<br>
 +
The beta subunit contains the metallocofactor (di-iron), required for the reduction initiation.
 +
<br>
  
 
==TSase==
 
==TSase==
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<br>
 
<br>
  
−
===Pfu variants===
+
==T7-LacO==
 +
 
 
<br>
 
<br>
−
Since error rate is important to be minimum in a number of techniques, there are several attempts to improve Pfu polymerase in that aspect with most importantly the Phusion variant [6]. Pfu can be attached to Sso7d which is a 7 kDa protein originating from Sulfolobus solfataricus, a hyperthermophilic archaebacterium [7]. Fusion to Sso7d has been proven to increase processivity, enabling longer amplifications and greater amplification speed [8].<br>
+
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.
 +
<br>
 +
 
 +
==mRFP1==
 +
 
 +
This protein is used to measure the expression of the transcriptional unit. The part that was used was BBa_E1010.
 +
 
 +
==RBS==
  
−
==Isolation and Purification==
 
 
<br>
 
<br>
−
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].
+
The RBS (BBa_B0030) was chosen from the existing parts of the iGEM Registry, due to its high popularity and efficiency for <i>E. coli</i>.
 
<br>
 
<br>
  
−
==Histidine Tag==
+
==Terminator==
 +
 
 
<br>
 
<br>
−
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).
+
The part that was used was taken from the iGEM Registry BBa_B0015.
 
<br>
 
<br>

Latest revision as of 17:52, 17 October 2021


pAdapted, a plasmid for the production of dNTP's

no

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE 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
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE 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
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE 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. The mRFP1 was added to measure the BBa_E1010.

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.

mRFP1

This protein is used to measure the expression of the transcriptional unit. The part that was used was BBa_E1010.

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.