Difference between revisions of "Part:BBa K4159009:Design"

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Description: This part consists of the AgrC and AgrA accessory regulatory gene sequences from the S. epidermidis bacteria that are a part of the quorum sensing gene complex. The part has been used in a bioreporter to detect AIP1 signaling molecules in the surroundings. The genes are separated by an RBS and spacer sequence and at the end, there is a double terminator. The AgrC and AgrA genes are derived from the basic parts BBa_K4159007 and BBa_K4159008 respectively. The ribosome binding site is from the common iGEM registry, B0034. The double-terminator in the end is also from the iGEM part registry B0015. In the middle there are some spacer nucleotides, but no specific restriction sites due to the fact that the composed part was ordered as one full fragment to make it easier during the iGEM project.  
 
Description: This part consists of the AgrC and AgrA accessory regulatory gene sequences from the S. epidermidis bacteria that are a part of the quorum sensing gene complex. The part has been used in a bioreporter to detect AIP1 signaling molecules in the surroundings. The genes are separated by an RBS and spacer sequence and at the end, there is a double terminator. The AgrC and AgrA genes are derived from the basic parts BBa_K4159007 and BBa_K4159008 respectively. The ribosome binding site is from the common iGEM registry, B0034. The double-terminator in the end is also from the iGEM part registry B0015. In the middle there are some spacer nucleotides, but no specific restriction sites due to the fact that the composed part was ordered as one full fragment to make it easier during the iGEM project.  
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Characterization: The part was cloned into plasmid pET28a-sfGFP by basic restriction site cloning.  The restriction sites used for cloning the part into the plasmid was XbaI and SphI. As the GFP encoding gene is already a part of the plasmid, we put this part in the opposite direction. However, with these restriction sites we also cut out the existing T7 promoter in the plasmid to replace with our own promoters, BBa_K4159011.  
 
Characterization: The part was cloned into plasmid pET28a-sfGFP by basic restriction site cloning.  The restriction sites used for cloning the part into the plasmid was XbaI and SphI. As the GFP encoding gene is already a part of the plasmid, we put this part in the opposite direction. However, with these restriction sites we also cut out the existing T7 promoter in the plasmid to replace with our own promoters, BBa_K4159011.  
  
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For ligation of the part, we used a rapid ligation kit from ThermoFisher, however we increased the ligation time up to 1h to ensure successful ligation. The part was successfully cloned. As this part was transformed into the same plasmid with BBa_K4159011  later on we wanted to verify that both parts were inserted correctly. We purified the plasmid from the cells and used both parts reverse and forward primer and ran a PCR. The gel run from the PCR shows great results of both parts being inserted. As both parts were inserted with specific sticky end restriction sites, we believe that both parts are also inserted the correct way.
 
  
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As the plasmid was purified from the cells, the cells also shifted in slight shade of green, which tells that the promoter part is inserted correctly and that the P2 promoter might be a little leaky even if it is not induced yet.
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[[File:BBa K415009 1.png|200px|thumb|left|BBa_K4159009 plasmid construct]]
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For ligation of the part, we used a rapid ligation kit from ThermoFisher, however we increased the ligation time up to 1h to ensure successful ligation. The part was successfully cloned (see fig. 1 and fig.2)
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[[File:BBa K415009 2.png|200px|thumb|left|Figure 1. 1 % agarose gel of the restriction digest of the pET28a-sfGFP plasmid and the BBa_K4159009 part, X presents an empty well. The marker [M] includes the GeneRulerTM ladder mix, ready-to-use with the base pairs [bp] indicated on the left side.
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]]
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[[File:BBa K415003 3.png|200px|thumb|left|Figure 2. 1 % agarose gel of the transformed and purified plasmid marked as P1, P2 and P3. Including a negative control (NC) and a positive control (PC). The marker [M] includes the GeneRulerTM ladder mix, ready-to-use with the base pairs [bp] indicated on the left side.
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]]
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As this part was transformed into the same plasmid with BBa_K4159011  later on we wanted to verify that both parts were inserted correctly. We purified the plasmid from the cells and used both parts reverse and forward primer and ran a PCR.
 +
 
 +
 
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[[File:BBa K415009 4.png|200px|thumb|left|Figure 4. Part 1 and part2 after amplification from the purified plasmid.
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]]
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Figure 4. shows great results of both parts being inserted. As both parts were inserted with specific sticky end restriction sites, we believe that both parts are also inserted the correct way.
 +
 
 +
As the plasmid was purified from the cells, the cells also shifted in slight shade of green, which tells that the promoter part is inserted correctly and that the P2 promoter might be a little leaky even if it is not induced yet.  
 +
 
 +
 
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References: [1]  Rutherford ST, Bassler BL. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med. 2012 Nov 1;2(11):a012427. doi: 10.1101/cshperspect.a012427. PMID: 23125205; PMCID: PMC3543102.
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===Source===
 
===Source===
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===References===
 
===References===
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Rutherford ST, Bassler BL. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med. 2012 Nov 1;2(11):a012427. doi: 10.1101/cshperspect.a012427. PMID: 23125205; PMCID: PMC3543102.

Latest revision as of 17:18, 1 October 2022


AgrC + AgrA (S.epidermidis)


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]


Design Notes

The AgrC and AgrA genes were codon optimized before ordering so that the complexity was tolerable and could be synthesized. These parts were ordered as one whole fragment as it was easier regarding the timeframe of the iGEM project.

Description: This part consists of the AgrC and AgrA accessory regulatory gene sequences from the S. epidermidis bacteria that are a part of the quorum sensing gene complex. The part has been used in a bioreporter to detect AIP1 signaling molecules in the surroundings. The genes are separated by an RBS and spacer sequence and at the end, there is a double terminator. The AgrC and AgrA genes are derived from the basic parts BBa_K4159007 and BBa_K4159008 respectively. The ribosome binding site is from the common iGEM registry, B0034. The double-terminator in the end is also from the iGEM part registry B0015. In the middle there are some spacer nucleotides, but no specific restriction sites due to the fact that the composed part was ordered as one full fragment to make it easier during the iGEM project. Characterization: The part was cloned into plasmid pET28a-sfGFP by basic restriction site cloning. The restriction sites used for cloning the part into the plasmid was XbaI and SphI. As the GFP encoding gene is already a part of the plasmid, we put this part in the opposite direction. However, with these restriction sites we also cut out the existing T7 promoter in the plasmid to replace with our own promoters, BBa_K4159011.


BBa_K4159009 plasmid construct

For ligation of the part, we used a rapid ligation kit from ThermoFisher, however we increased the ligation time up to 1h to ensure successful ligation. The part was successfully cloned (see fig. 1 and fig.2)


Figure 1. 1 % agarose gel of the restriction digest of the pET28a-sfGFP plasmid and the BBa_K4159009 part, X presents an empty well. The marker [M] includes the GeneRulerTM ladder mix, ready-to-use with the base pairs [bp] indicated on the left side.


Figure 2. 1 % agarose gel of the transformed and purified plasmid marked as P1, P2 and P3. Including a negative control (NC) and a positive control (PC). The marker [M] includes the GeneRulerTM ladder mix, ready-to-use with the base pairs [bp] indicated on the left side.


As this part was transformed into the same plasmid with BBa_K4159011 later on we wanted to verify that both parts were inserted correctly. We purified the plasmid from the cells and used both parts reverse and forward primer and ran a PCR.


Figure 4. Part 1 and part2 after amplification from the purified plasmid.


Figure 4. shows great results of both parts being inserted. As both parts were inserted with specific sticky end restriction sites, we believe that both parts are also inserted the correct way.

As the plasmid was purified from the cells, the cells also shifted in slight shade of green, which tells that the promoter part is inserted correctly and that the P2 promoter might be a little leaky even if it is not induced yet.


References: [1] Rutherford ST, Bassler BL. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med. 2012 Nov 1;2(11):a012427. doi: 10.1101/cshperspect.a012427. PMID: 23125205; PMCID: PMC3543102.


Source

[1] https://www.genome.jp/entry/ser:SERP1493 [2] https://www.genome.jp/entry/ser:SERP1492


References

Rutherford ST, Bassler BL. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med. 2012 Nov 1;2(11):a012427. doi: 10.1101/cshperspect.a012427. PMID: 23125205; PMCID: PMC3543102.