Difference between revisions of "Part:BBa K4075013"

 
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'''Figure 3.''' The first lane is a positive control, with the PCR product from amplification oh this par synthesis (gBlock). A linear sample of pBBR1MCS-2 was used as a negative control. On the third lane, the G0 assembly on <i>E. coli</i> DH5-alpha was confirmed as positive.  
 
'''Figure 3.''' The first lane is a positive control, with the PCR product from amplification oh this par synthesis (gBlock). A linear sample of pBBR1MCS-2 was used as a negative control. On the third lane, the G0 assembly on <i>E. coli</i> DH5-alpha was confirmed as positive.  
  
Sequence and Features
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4075013 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K4075013 parameters</partinfo>
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Latest revision as of 03:15, 22 October 2021


T7-LacO Promoter + RiboJ + RBS_0034 + mCherry + double Terminator


This part is made of a T7-LacO Promoter, Ribo J, RBS_0034, OmpA signal peptide, 6xHis-tag, followed by the sequence coding for the reporter gene mCherry, and ends in a double terminator. This device enables the expression of the fluorescent protein mCherry in Escherichia coli strains with T7 polymerase.

Assembly

The synthesis of the device T7-LacO Promoter + RiboJ + RBS_0034 + mCherry + double Terminator plus overlaps insert and linear pBBR1MCS-2 plasmids were incubated with Gibson Assembly Master Mix at 50°C for 1 hour. The assembly mixture was then transformed into competent E.coli DH5-alpha cells by heat shock at 42°C for 10 seconds and plated onto Luria Broth Agar plates supplemented with Kanamycin at working concentration for selection. The isolated colonies were cultivated and a miniprep was performed.

A PCR from miniprep plasmid, under the following conditions: 98°C for 40 seconds, 30X cycles of: 98°C for 10 seconds, 65°C for 30 seconds, 72°C for 35 seconds. Final extension at 72°C for 2 minutes. PCR product held at 4°. PCR products were run on a 0,8% agarose gel at 50 V for 45 minutes and visualised in a transilluminator setting. Primers used at the overlap section:

Whole insert Forward :

  • 5’-GTGCTGCAAGGCGATTAAGTTG

Whole insert Reverse :

  • 5’-TTACAACGTCGTGACTGGGAAC

T--UNILA Latam--Result3.png

Figure 3. The first lane is a positive control, with the PCR product from amplification oh this par synthesis (gBlock). A linear sample of pBBR1MCS-2 was used as a negative control. On the third lane, the G0 assembly on E. coli DH5-alpha was confirmed as positive.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]