Difference between revisions of "Part:BBa K4147010"
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===Characterization of RFP construct with LacI regulated promoter for bacterial expression=== | ===Characterization of RFP construct with LacI regulated promoter for bacterial expression=== | ||
| + | <p align="justify"> | ||
| + | Before moving on to the experimental process, we first performed an <i>in silico</i> analysis in SnapGene®️ to simulate our ligated expression plasmid. The theoretical result was a sequence of 3,286 bp, as shown in <b>Figure 1.</b></p> | ||
| + | |||
<center>[[File:MCherry-Tec-Chihuahua.png |300px]]</center> | <center>[[File:MCherry-Tec-Chihuahua.png |300px]]</center> | ||
<center><b>Figure 1</b>. SnapGene®️ map of BioBrick BBa_K4147010.</center> | <center><b>Figure 1</b>. SnapGene®️ map of BioBrick BBa_K4147010.</center> | ||
| + | <p align="justify"> | ||
| + | Our insert of PcOSM Construct with LacI regulated promoter was later synthetized by Twist Bioscience with the Biobrick prefix and suffix as well as adapters flanking the composite part for easiest restriction digest. EcoRI and PstI enzymes were used to digest both construct and vector. Once digested we proceed to ligate our insert into a pTwist Kan High Copy plasmid with Anza™ T4 DNA Ligase Master Mix. The ligation product was then transformed into <i>E. coli</i> BL21(DE3) cells by heat shock.</p> | ||
| + | |||
| + | <p align="justify"> | ||
| + | The next step was to confirm the presence of the vector of interest in our chassis after transformation, so we performed colony PCR using Forward: 5'-GTTTCTTCGAATTCGCGGCCGCTTCTA and Reverse: 5'-GTTTCTTCCTTCCTGCAGCGGCCGCTACTAG primers specific for the BioBrick prefix and suffix respectively. The PCR action from SnapGene®️ was used to predict the resultant agarose gel.</p> | ||
| + | |||
| + | <center>[[File:Agarose-gel-rfp-laci.png |400px]]</center> | ||
| + | <center><b>Figure 2</b>.<i>Left.</i> SnapGene®️ amplification through PCR of BBa_K4147003 on an 0.8% agarose gel with NEB Quick-Load Purple 1Kb Plus DNA Ladder. <i>Right</i> D) Amplification of PcOSM Construct with LacI regulated promoter where the highlighted band corresponds to approximately 1,137 bp.</center> | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K4147010 parameters</partinfo> | <partinfo>BBa_K4147010 parameters</partinfo> | ||
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Revision as of 05:44, 10 October 2022
mCherry (RFP) construct with LacI regulated promoter for bacterial expression
This mCherry reporter is one of several "second-generation" monomeric fluorescent proteins. The proposed construct for its expression it's conformed by a LacI regulated promoter (BBa_R0010), an RBS (BBa_B0032) and a double terminator (BBa_B0015). Lastly this construct was optimized for its production on E. coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization of RFP construct with LacI regulated promoter for bacterial expression
Before moving on to the experimental process, we first performed an in silico analysis in SnapGene®️ to simulate our ligated expression plasmid. The theoretical result was a sequence of 3,286 bp, as shown in Figure 1.
Our insert of PcOSM Construct with LacI regulated promoter was later synthetized by Twist Bioscience with the Biobrick prefix and suffix as well as adapters flanking the composite part for easiest restriction digest. EcoRI and PstI enzymes were used to digest both construct and vector. Once digested we proceed to ligate our insert into a pTwist Kan High Copy plasmid with Anza™ T4 DNA Ligase Master Mix. The ligation product was then transformed into E. coli BL21(DE3) cells by heat shock.
The next step was to confirm the presence of the vector of interest in our chassis after transformation, so we performed colony PCR using Forward: 5'-GTTTCTTCGAATTCGCGGCCGCTTCTA and Reverse: 5'-GTTTCTTCCTTCCTGCAGCGGCCGCTACTAG primers specific for the BioBrick prefix and suffix respectively. The PCR action from SnapGene®️ was used to predict the resultant agarose gel.
