Difference between revisions of "Part:BBa K3352008"

 
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<partinfo>BBa_K3352008 short</partinfo>
 
<partinfo>BBa_K3352008 short</partinfo>
  
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Construct Design
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The composite part utilizes a T7 Promoter (BBa_J64997), extended RBS (BBa_K3352002), SplintR Ligase (BBa_K3352000), and a T7 Terminator (BBa_K3352003).
  
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We improved this construct by using pET3a vectors with appropriate BioBrick prefixes and suffixes that fulfill the assembly standard. pET vectors include the T7 bacteriophage gene 10, which promotes high-level transcription and translation. Utilizing both a T7 promoter, T7 terminator, and an extended UTR sequence around the RBS and before the terminator, we would maximize the protein expression for our enzymes.
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<b><font size="+1.2"> Construct Design </font></b>
  
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Characterization
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We improved the constructs (BBa_K3352006) and (BBa_K33352007) by using pET3a and pET11a vectors with appropriate BioBrick prefixes and suffixes that fulfill the assembly standard. The pET vector includes the T7 promoter, which promotes high level transcription. By utilizing both a T7 promoter, T7 terminator, and an extended UTR sequence around the RBS and before the terminator, we would maximize the protein expression for our enzymes. These composite parts were synthesized by GenScript.
  
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Our SDS-PAGE results show that both purified proteins migrate at the expected sizes. However, due to the presence of unfavorable buffer conditions in our eluted proteins, we performed a buffer exchange to reach the desired pH and storage conditions for our enzymes. We used these proteins for our viral detection test.  
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https://2020.igem.org/wiki/images/6/61/T--TAS_Taipei--Parts_Extended_RBS.png
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<b> Figure 1: Extended RBS </b>
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<b><font size="+1.2"> Characterization </font></b>
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<b><font size="+0.5"> pET3a and pET11a Promoter with T7 Terminator </font></b>
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We also aimed to improve this construct by using a pET3a and pET11a vectors with appropriate BioBrick prefixes and suffixes that fulfill the assembly standard. pET vectors include the T7 promoter, which promotes high level transcription [1]. By utilizing both a T7 promoter, T7 terminator, and an extended UTR sequence around the RBS and before the terminator, we can maximize protein expression for our enzymes (Figure 2) [1]. These composite parts were synthesized by GenScript.
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https://2020.igem.org/wiki/images/7/78/T--TAS_Taipei--Registry_13.png
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<b> Figure 2: Characterization of the pET T7 promoter with Φ29 polymerase (BBa_K3352009) construct and pET T7 promoter with SplintR (BBa_K3352008). We digested both constructs at the XbaI and PstI sites. The part (BBa_K3352001) was ligated into a pET11a backbone that is about 5500bps to form part (BBa_K3352009). Similarly, the part (BBa_K3352000) was ligated into pET3a backbone with a size of 4400bps which forms part (BBa_K3352008). </b>
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<b><font size="+0.5"> Protein Expression and Purification </font></b>
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We transformed our plasmids into BL21(DE3) <i>E. coli</i> cells. We grew bacterial cultures overnight at 37°C, diluted them to an OD600 of 0.2, and then grew them to 0.5, where we collected a sample of 1mL. We then added IPTG and grew the cultures for another 4 hours. After the additional 4 hours, we then collected another 1mL sample. We centrifuged both samples and resuspended the pellets in 1x Sample Buffer. The samples containing IPTG expressed the protein more strongly, which suggests that our protein was present (Figure 3).
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https://2020.igem.org/wiki/images/b/b8/T--TAS_Taipei--Registry_8.png
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<b> Figure 3: SDS-PAGE results show that SplintR ligase and Φ29 polymerase was expressed by <i>E. coli</i>. We grew the bacterial cultures overnight at 37°C, diluted them to an OD600 of 0.2, and then grew them to 0.5, where we collected a sample of 1mL. We then added IPTG and grew the cultures for another 4 hours. After the additional 4 hours, we then collected another 1mL sample. We centrifuged both samples and resuspended the pellets in 1x Sample Buffer. The sample with the IPTG expressed the protein more strongly, which suggests that our protein was present at 68.2 kDa. </b>
  
  

Revision as of 12:26, 25 October 2020


T7 + New RBS & Terminator + SplintR Ligase Expressing Construct

The composite part utilizes a T7 Promoter (BBa_J64997), extended RBS (BBa_K3352002), SplintR Ligase (BBa_K3352000), and a T7 Terminator (BBa_K3352003).

Construct Design

We improved the constructs (BBa_K3352006) and (BBa_K33352007) by using pET3a and pET11a vectors with appropriate BioBrick prefixes and suffixes that fulfill the assembly standard. The pET vector includes the T7 promoter, which promotes high level transcription. By utilizing both a T7 promoter, T7 terminator, and an extended UTR sequence around the RBS and before the terminator, we would maximize the protein expression for our enzymes. These composite parts were synthesized by GenScript.


T--TAS_Taipei--Parts_Extended_RBS.png

Figure 1: Extended RBS


Characterization


pET3a and pET11a Promoter with T7 Terminator

We also aimed to improve this construct by using a pET3a and pET11a vectors with appropriate BioBrick prefixes and suffixes that fulfill the assembly standard. pET vectors include the T7 promoter, which promotes high level transcription [1]. By utilizing both a T7 promoter, T7 terminator, and an extended UTR sequence around the RBS and before the terminator, we can maximize protein expression for our enzymes (Figure 2) [1]. These composite parts were synthesized by GenScript.

T--TAS_Taipei--Registry_13.png

Figure 2: Characterization of the pET T7 promoter with Φ29 polymerase (BBa_K3352009) construct and pET T7 promoter with SplintR (BBa_K3352008). We digested both constructs at the XbaI and PstI sites. The part (BBa_K3352001) was ligated into a pET11a backbone that is about 5500bps to form part (BBa_K3352009). Similarly, the part (BBa_K3352000) was ligated into pET3a backbone with a size of 4400bps which forms part (BBa_K3352008).


Protein Expression and Purification

We transformed our plasmids into BL21(DE3) E. coli cells. We grew bacterial cultures overnight at 37°C, diluted them to an OD600 of 0.2, and then grew them to 0.5, where we collected a sample of 1mL. We then added IPTG and grew the cultures for another 4 hours. After the additional 4 hours, we then collected another 1mL sample. We centrifuged both samples and resuspended the pellets in 1x Sample Buffer. The samples containing IPTG expressed the protein more strongly, which suggests that our protein was present (Figure 3).

T--TAS_Taipei--Registry_8.png

Figure 3: SDS-PAGE results show that SplintR ligase and Φ29 polymerase was expressed by E. coli. We grew the bacterial cultures overnight at 37°C, diluted them to an OD600 of 0.2, and then grew them to 0.5, where we collected a sample of 1mL. We then added IPTG and grew the cultures for another 4 hours. After the additional 4 hours, we then collected another 1mL sample. We centrifuged both samples and resuspended the pellets in 1x Sample Buffer. The sample with the IPTG expressed the protein more strongly, which suggests that our protein was present at 68.2 kDa.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1029
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 869
  • 1000
    COMPATIBLE WITH RFC[1000]