Difference between revisions of "Part:BBa K4012003"

 
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<partinfo>BBa_K4012003 parameters</partinfo>
 
<partinfo>BBa_K4012003 parameters</partinfo>
 
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===Obtaining the pPGK1 fragment and BsaI digested verification===
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[[Image:T--AISSU_Union--partslast.jpg|thumbnail|750px|center|'''Figure 1:'''
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[https://parts.igem.org/Part:BBa_K4012003] Results of yeast toolkit plasmids enzyme-digested verification]]
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We construct pGAL1 with vector type8-pSB1K3-GFP and proceed digested verification using BsaI.According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments Type-4-tPGK1(238bp) matched with previous expectations by compared with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.
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===pPGK1 in Level1 plasmid assembly===
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[[Image:4CL.jpg|thumbnail|750px|center|'''Figure 2:'''
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[https://parts.igem.org/Part:BBa_K4012003] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence Pc4CL]]
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The construction schematic of CrCPR sequence demonstrated as Fig.2. The initiation of the CrCPR sequence is done by promoter pGAL1, with termination done by tTDH1. The sequence ConLS and ConR1 are connector sequences within the Level 1 plasmid assembly. Furthermore, typr9-KVF and type9-VR are imposed to enact selection through colonies PCR, results shown in Fig.7 The band length 3300bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.
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===pPGK1 in Level2 plasmid assembly===
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[[Image:yeast PCR.jpg|thumbnail|750px|center|'''Figure 3:'''
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[https://parts.igem.org/Part:BBa_K4012003] A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway]]
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pGAL1 is also involved in assembly of synthesis pathway of naringenin, shown by Fig.3.

Revision as of 10:53, 21 October 2021


pPGK1

A Saccharomyces cerevisiae promoter. It is used to initiate MsCHI sequence to express

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 1105
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3
    Illegal BsaI.rc site found at 1115


Obtaining the pPGK1 fragment and BsaI digested verification

Figure 1: [1] Results of yeast toolkit plasmids enzyme-digested verification

We construct pGAL1 with vector type8-pSB1K3-GFP and proceed digested verification using BsaI.According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments Type-4-tPGK1(238bp) matched with previous expectations by compared with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

pPGK1 in Level1 plasmid assembly

Figure 2: [2] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence Pc4CL

The construction schematic of CrCPR sequence demonstrated as Fig.2. The initiation of the CrCPR sequence is done by promoter pGAL1, with termination done by tTDH1. The sequence ConLS and ConR1 are connector sequences within the Level 1 plasmid assembly. Furthermore, typr9-KVF and type9-VR are imposed to enact selection through colonies PCR, results shown in Fig.7 The band length 3300bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

pPGK1 in Level2 plasmid assembly

Figure 3: [3] A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway

pGAL1 is also involved in assembly of synthesis pathway of naringenin, shown by Fig.3.