Difference between revisions of "Part:BBa K3257042"
 
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''When the organization has once begun to vary, it generally continues varying for many generations.'' – Charles Darwin ''The Origin of Life''
 
''When the organization has once begun to vary, it generally continues varying for many generations.'' – Charles Darwin ''The Origin of Life''
  
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We would like to present this part as our best basic part. Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is responsible for the reverse transcription process of the target Gene of Interest (GOI), which is rather one of the most important processes in our system.
+
We would like to present this part as our best basic part.  
 +
Naturally, when Moloney Murine Leukemia Virus (MMLV) infects its host, gag-pol polyprotein encoded in the MMLV genome is expressed. This polyprotein will be further cleaved into the capsid protein (about 60.4kDa) and the reverse transcriptase (about 69.1kDa) by a protease (about 13.5 kDa) also encoded in the polyprotein mRNA (Figure 1).
  
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MMLV RT acts as a reverse transcription tool with a relatively low fidelity so that it is qualified for a random mutation generator. Also, a previous study showed that point mutation (nucleotide from A to T) of at the 586th amino acid residual (from Y to F) of the polymerase itself (encoded merely by pol gene) (BBa_K3257043 https://parts.igem.org/Part:BBa_K3257043) further improves its error-prone nature by ≈5-fold in a single replication cycle (please refer to https://parts.igem.org/Part:BBa_K3257043).
+
[[File:Gag-pol.png|center|300px|thumb|'''Figure 1. Exhibition of gene encoding gag-pol polyprotein''']]
 +
 
 +
Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is responsible for the reverse transcription process of the target gene, which is rather one of the most important processes in our system.
 +
 
 +
MMLV RT acts as a reverse transcription tool with a relatively low fidelity so that it is qualified for a random mutation generator. Also, a previous study showed that point mutation (nucleotide from A to T) of at the 586th amino acid residual (from Y to F) of the polymerase itself (encoded merely by pol gene) (BBa_K3257043 https://parts.igem.org/Part:BBa_K3257043) further improves its error-prone nature by ≈5-fold in a single reverse transcription cycle (please refer to https://parts.igem.org/Part:BBa_K3257043).
  
 
MMLV RT was first designed to be transcribed into a single mRNA with a stop codon between the gag gene (encoding capsid protein) and pol gene (encoding the reverse transcriptase), which could not be properly expressed ''in vivo'' (of ''E.coli'') when we fused an EGFP after the gag-pol CDS.
 
MMLV RT was first designed to be transcribed into a single mRNA with a stop codon between the gag gene (encoding capsid protein) and pol gene (encoding the reverse transcriptase), which could not be properly expressed ''in vivo'' (of ''E.coli'') when we fused an EGFP after the gag-pol CDS.
  
−
Frustratedly, we set out to solve the problem by cloning it into various kinds of vectors, after different promoters and mutating the stop codon between gag and pol into Glutamine (from T to C), an intermediate amino acid residue as a read-through. (In wild type Moloney Murine Leukemia virus, there is a stop codon between mRNA transcribed from the gag gene and pol gene while it can be read through as a Glutamine when translated by ribosome to form gag and gag-pol polyprotein at a ratio of 20:1. We have changed the stop codon into a Glutamine codon to assure its readability in ''E.coli''.)
+
Frustratedly, we set out to solve the problem by cloning it into various kinds of vectors, after different promoters and mutating the stop codon between gag and pol into Glutamine (from T to C), an intermediate amino acid residue as a read-through. (In wild type Moloney Murine Leukemia Virus, there is a stop codon between mRNA transcribed from the gag gene and pol gene while it can be read through as a Glutamine when translated by ribosome to form gag and gag-pol polyprotein at a ratio of 20:1. We have changed the stop codon into a Glutamine codon to assure its readability in ''E.coli''.)
 +
 
 +
Using SDS-PAGE, we have verified its expression and self-cleavage in ''E.coli'' (Figure 2).
 +
 
 +
[[File:Gag-pol-sds page.png|center|500px|thumb|'''Figure 2. SDS-PAGE of gag-pol polyprotein expression''' This SDS-PAGE analysis proves the expression and self-cleavage of gag-pol polyprotein. As displayed in the result, WT represents the gag-Q-pol polyprotein (BBa_K3257042 https://parts.igem.org/Part:BBa_K3257042) and Mutant represents the Y586F mutant of gag-Q-pol polyprotein (BBa_K3257043 https://parts.igem.org/Part:BBa_K3257043). - represents the uninduced bacteria culture sample and + represents the induced bacteria culture. Comparing + and -, we can see that IPTG induction enables protease (about 13.5 kDa) to be expressed and we can also see an increase of capsid protein (about 60.4 kDa) and reverse transcriptase (about 69.1 kDa) expression.]]
  
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Using SDS-PAGE and qRT-PCR, we have verified its expression in ''E.coli'' (Figure 1a) and its function as a reverse transcriptase (Figure 1b). Furthermore, its mutation rate is able to be measured by high throughput sequencing. In the future, this part can be used to express gag-pol polyprotein inside the bacteria to initiate the reverse transcription process ''in vivo''.
+
Furthermore, its mutation rate is able to be measured by high throughput sequencing. In the future, this part can be used to express gag-pol polyprotein inside the bacteria to initiate the reverse transcription process ''in vivo''.
  
  

Latest revision as of 19:13, 21 October 2019

MMLV gag-pol Polyprotein

When the organization has once begun to vary, it generally continues varying for many generations. – Charles Darwin The Origin of Life

We would like to present this part as our best basic part. Naturally, when Moloney Murine Leukemia Virus (MMLV) infects its host, gag-pol polyprotein encoded in the MMLV genome is expressed. This polyprotein will be further cleaved into the capsid protein (about 60.4kDa) and the reverse transcriptase (about 69.1kDa) by a protease (about 13.5 kDa) also encoded in the polyprotein mRNA (Figure 1).

Figure 1. Exhibition of gene encoding gag-pol polyprotein

Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is responsible for the reverse transcription process of the target gene, which is rather one of the most important processes in our system.

MMLV RT acts as a reverse transcription tool with a relatively low fidelity so that it is qualified for a random mutation generator. Also, a previous study showed that point mutation (nucleotide from A to T) of at the 586th amino acid residual (from Y to F) of the polymerase itself (encoded merely by pol gene) (BBa_K3257043 https://parts.igem.org/Part:BBa_K3257043) further improves its error-prone nature by ≈5-fold in a single reverse transcription cycle (please refer to https://parts.igem.org/Part:BBa_K3257043).

MMLV RT was first designed to be transcribed into a single mRNA with a stop codon between the gag gene (encoding capsid protein) and pol gene (encoding the reverse transcriptase), which could not be properly expressed in vivo (of E.coli) when we fused an EGFP after the gag-pol CDS.

Frustratedly, we set out to solve the problem by cloning it into various kinds of vectors, after different promoters and mutating the stop codon between gag and pol into Glutamine (from T to C), an intermediate amino acid residue as a read-through. (In wild type Moloney Murine Leukemia Virus, there is a stop codon between mRNA transcribed from the gag gene and pol gene while it can be read through as a Glutamine when translated by ribosome to form gag and gag-pol polyprotein at a ratio of 20:1. We have changed the stop codon into a Glutamine codon to assure its readability in E.coli.)

Using SDS-PAGE, we have verified its expression and self-cleavage in E.coli (Figure 2).

Figure 2. SDS-PAGE of gag-pol polyprotein expression This SDS-PAGE analysis proves the expression and self-cleavage of gag-pol polyprotein. As displayed in the result, WT represents the gag-Q-pol polyprotein (BBa_K3257042 https://parts.igem.org/Part:BBa_K3257042) and Mutant represents the Y586F mutant of gag-Q-pol polyprotein (BBa_K3257043 https://parts.igem.org/Part:BBa_K3257043). - represents the uninduced bacteria culture sample and + represents the induced bacteria culture. Comparing + and -, we can see that IPTG induction enables protease (about 13.5 kDa) to be expressed and we can also see an increase of capsid protein (about 60.4 kDa) and reverse transcriptase (about 69.1 kDa) expression.

Furthermore, its mutation rate is able to be measured by high throughput sequencing. In the future, this part can be used to express gag-pol polyprotein inside the bacteria to initiate the reverse transcription process in vivo.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 174
    Illegal BglII site found at 1286
    Illegal BglII site found at 2592
    Illegal BglII site found at 3824
    Illegal BamHI site found at 2609
    Illegal BamHI site found at 2915
    Illegal XhoI site found at 940
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1825
    Illegal AgeI site found at 3396
  • 1000
    COMPATIBLE WITH RFC[1000]