Difference between revisions of "Part:BBa K4887022"
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4887022 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4887022 SequenceAndFeatures</partinfo>
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<h1>13.5 Results:</h1>
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<h2>(1) Construction of the expression vector of sgRNA (IbGBSSI)</h2>
 +
The validated backbone vector of sgRNA (IbGBSSI) was digested by EcoR I & Hind III and inserted into the corresponding sites of the binary vector pCAMBIA1301s, harbouring the Hygromycin B resistance gene HygR and the reporting gene GUS, and obtained the expression vector of sgRNA (IbGBSSI): psgR-Cas9-sgRNA(IbGBSSI)-p1301s.
 +
 +
<h2>(2) Agrobacterium tumefaciens transformation</h2>
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The expression vector of sgRNA (IbGBSSI) was then transferred into Agrobacterium tumefaciens LB4404 by reeze-thaw method. The positive transformants containing were selected and cultured on solid TY medium plates containing antibiotics of spectinomycin and kanamycin <b class="red">(Fig. 1)</b>. The obtained clones were validated by performing PCR detection for the sgRNA sequence with primers M13F/oligo2 (The sequence of M11F was: 5’-TGTAAAACGA CGGCCAGT-3’). The gel electrophoresis results <b class="red">(Fig. 2)</b> showed that the gene band were approximately 100bp, as expected, indicating that expression vector of sgRNA (IbGBSSI) was been constructed and transferred into Agrobacterium tumefaciens successfully.
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<div class="center">
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<img class="bild toobig" src="https://static.igem.wiki/teams/4887/wiki/images/images/part-a-tumefaciens-transformed-with-the-vector-psgr-cas9-sgrna-ibgbssi-p1301s.jpg" />
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<div class="unterschrift">
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<b>Fig. 1 A. tumefaciens transformed with the vector psgR-Cas9-sgRNA(IbGBSSI)-p1301s</b>
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<div class="center">
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<img class="bild" src="https://static.igem.wiki/teams/4887/wiki/images/images/part-pcr-result-of-sgrna-ibgbssi-in-a-tumefaciens-transformed-with-expression-vector-of-sgrna-ibgbssi.jpg" />
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<div class="unterschrift">
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<b>
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Fig. 2 PCR result of sgRNA (IbGBSSI) in A. tumefaciens transformed with expression vector of sgRNA (IbGBSSI)
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</b>
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</div>
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</div>
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<h2>(3) Genetic transformation of sweet potato</h2>
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Embryogenic calli of sweet potato ware infected with the A. tumefaciens transformants containing expression vector of sgRNA (IbGBSSI) by co-culture on the MSD media containing hygromycin B and cefalexin <b class="red">(Fig. 3-A)</b>. After selection with hygromycin B, positive transformed calluses were obtained <b class="red">(Fig. 3-B)</b>. These calli were further cultured to obtain transgenic sweet potato seedlings <b class="red">(Fig. 4)</b>.
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<div class="center">
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<img class="bild" src="https://static.igem.wiki/teams/4887/wiki/images/images/part-embryogenic-callus-co-culture-with-a-tumefaciens-transformants.png" />
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<div class="unterschrift">
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<b>
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Fig. 3 Embryogenic callus co-culture with A. tumefaciens transformants (A) and selection of postively transformed callus with hygromycin (B)
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</b>
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</div>
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</div>
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<div class="center">
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<img class="bild toobig" src="https://static.igem.wiki/teams/4887/wiki/images/images/part-seedlings-of-ibgbssi-knockout-lines-after-preliminary-selections.jpg" />
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<div class="unterschrift">
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<b>
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Fig. 4 Seedlings of IbGBSSI-knockout lines after preliminary selections
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</b>
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</div>
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</div>
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<h2>(4) Verification of transgenic sweet potato plants</h2>
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<h3>1) GUS detection</h3>
 +
The GUS gene, is a commonly used reporter gene harboured in pCAMBIA1301s. Its expression product β-glucuronidase is a hydrolase that can catalyze the hydrolysis of many β-glucoside esters. It can decompose X-Gluc into blue substances, to observe the expression of foreign genes in transgenic plants and identify transgenic plants.
 +
<br />
 +
After the regenerated seedlings grew leaves, GUS staining was performed on these transgenic plants. Two successfully transformed sweet potato lines were preliminarily screened and designated as 23216004 and 23216005, whose leaves turned green <b class="red">(Fig. 5)</b>.
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<div class="center">
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<img class="bild" src="https://static.igem.wiki/teams/4887/wiki/images/images/part-results-of-gus-staining.png" />
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<div class="unterschrift">
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<b>
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Fig. 5 Results of GUS staining
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</b>
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</div>
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</div>
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<h3>2) PCR detection </h3>
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Genome DNA of these two transgenic lines was extracted from leaves of these two regenerated seedlings. PCR detection on the genomes was performed by using two pairs of primers which were designed based on the Cas9 protein gene and the hygromycin resistance gene (HygR), respectively. As the result, the transgenic lines 23216004 and 23216005 were further validated <b class="red">(Fig. 6)</b>.<br />
 +
The sequences of the two pairs of primers were as bellow:
 +
<ul>
 +
<li>CAS9-F: 5’-atggactataaggaccacgacgg-3’; CAS9-R: 5’-ttgtcgcctcccagctgagacag-3’</li>
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<li>HygR-F: 5’-Atgaaaaagcctgaactcac-3’;    HygR-R: 5’-ctatttctttgccctcggac-3’</li>
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</ul> <br />
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Subsequently, these two transgenic lines and the wild-type B23 were planted with the method of cuttage in an experimental greenhouse to harvest starch-rich tubers.
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<div class="center divbig">
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<img class="bild toobig two" src="https://static.igem.wiki/teams/4887/wiki/images/images/part-detection-results-of-pcrs-for-gene-hygr.png" />
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<img class="bild toobig two" src="https://static.igem.wiki/teams/4887/wiki/images/images/part-detection-results-of-pcrs-for-gene-cas9.png" />
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<div class="unterschrift">
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<b>
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Fig. 6 Detection results of PCRs for gene HygR and gene Cas9.
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</b>
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</div>
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</div>
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<h3>3) Determination of the expression level of IbGBSSI in root tubers</h3>
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Two months after transplantation, the tubers of the transgenic lines were harvested. It showed that the number and size of the root tubers were largely consistent with the wild type <b class="red">(Fig. 7)</b>.
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 +
<div class="center">
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<img class="bild" src="https://static.igem.wiki/teams/4887/wiki/images/images/part-phenotypes-of-the-ibgbssi-knockout-lines-planted-in-greenhouse.png" />
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<div class="unterschrift">
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<b>
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Fig. 7 Phenotypes of the IbGBSSI-knockout lines planted in greenhouse
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</b>
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</div>
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</div>
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RNA was extracted from the fresh root tubers and cDNA was obtained through reverse transcription later. Then, the relative expression level of the gene IbGBSSI was determined with the method of Quantitative Real-time PCR with the root-tuber cDNA as templates. The result showed that the relative expression level of IbGBSSI in root tubers of the transgenic lines (0.1063 and 0.2407) was much lower than that of the wild type (1.0000) <b class="red">(Fig. 8)</b>. It revealed that the knock-out of IbGBSSI in the pathway of starch synthesis was successful.
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 +
<div class="center">
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<img class="bild toobig" src="https://static.igem.wiki/teams/4887/wiki/images/images/part-q-pcr-result-of-the-relative-expression-level-of-ibgbssi-in-root-tubers.png" />
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<div class="unterschrift">
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<b>
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Fig. 8 Q-PCR result of the relative expression level of IbGBSSI in root tubers
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</b>
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</div>
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</div>
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<h3>(5) Starch analysis of transgenic sweet potato root tubers<h3>
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Freshly harvested sweet potato tubers were cleaned, peeled, and sliced into small pieces. Starch was extracted from these pieces for qualitative and quantitative detection afterwards.<br />
 +
<h4>1) Qualitative detection of the starch components <h4>
 +
When exposed to iodine, Amylose appears blue, while amylopectin appears reddish brown or purple red. Therefore, the component qualitative detection of the total starch from the transgenic lines was performed. As the result, the total starch of the transgenic lines appears reddish brown while that appears blue of the wild type <b class="red">(Fig. 9)</b>. It indicated that the total starch of the transgenic lines was composed mainly of amylopectin.
 +
<div class="center">
 +
<img class="bild" src="https://static.igem.wiki/teams/4887/wiki/images/images/part-component-detection-of-total-starch-by-iodine-staining.png" />
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<div class="unterschrift">
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<b>
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Fig. 9 Component detection of total starch by iodine staining
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</b>
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Revision as of 11:09, 9 October 2023


Expression vector of IbGBSSI knockout system

This part is the expression vector used for knockout gene IbGASSI (BBa_K4887001) in sweet potato (Ipomoea batatas).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 2106
    Illegal PstI site found at 3528
    Illegal PstI site found at 3732
    Illegal PstI site found at 3762
    Illegal PstI site found at 4974
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 2106
    Illegal PstI site found at 3528
    Illegal PstI site found at 3732
    Illegal PstI site found at 3762
    Illegal PstI site found at 4974
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1567
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 2106
    Illegal PstI site found at 3528
    Illegal PstI site found at 3732
    Illegal PstI site found at 3762
    Illegal PstI site found at 4974
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 2106
    Illegal PstI site found at 3528
    Illegal PstI site found at 3732
    Illegal PstI site found at 3762
    Illegal PstI site found at 4974
    Illegal NgoMIV site found at 1157
    Illegal NgoMIV site found at 1176
    Illegal NgoMIV site found at 2394
    Illegal NgoMIV site found at 3498
    Illegal NgoMIV site found at 3571
    Illegal NgoMIV site found at 4056
    Illegal NgoMIV site found at 4965
    Illegal AgeI site found at 6940
  • 1000
    COMPATIBLE WITH RFC[1000]

13.5 Results:

(1) Construction of the expression vector of sgRNA (IbGBSSI)

The validated backbone vector of sgRNA (IbGBSSI) was digested by EcoR I & Hind III and inserted into the corresponding sites of the binary vector pCAMBIA1301s, harbouring the Hygromycin B resistance gene HygR and the reporting gene GUS, and obtained the expression vector of sgRNA (IbGBSSI): psgR-Cas9-sgRNA(IbGBSSI)-p1301s.

(2) Agrobacterium tumefaciens transformation

The expression vector of sgRNA (IbGBSSI) was then transferred into Agrobacterium tumefaciens LB4404 by reeze-thaw method. The positive transformants containing were selected and cultured on solid TY medium plates containing antibiotics of spectinomycin and kanamycin (Fig. 1). The obtained clones were validated by performing PCR detection for the sgRNA sequence with primers M13F/oligo2 (The sequence of M11F was: 5’-TGTAAAACGA CGGCCAGT-3’). The gel electrophoresis results (Fig. 2) showed that the gene band were approximately 100bp, as expected, indicating that expression vector of sgRNA (IbGBSSI) was been constructed and transferred into Agrobacterium tumefaciens successfully.
Fig. 1 A. tumefaciens transformed with the vector psgR-Cas9-sgRNA(IbGBSSI)-p1301s
Fig. 2 PCR result of sgRNA (IbGBSSI) in A. tumefaciens transformed with expression vector of sgRNA (IbGBSSI)

(3) Genetic transformation of sweet potato

Embryogenic calli of sweet potato ware infected with the A. tumefaciens transformants containing expression vector of sgRNA (IbGBSSI) by co-culture on the MSD media containing hygromycin B and cefalexin (Fig. 3-A). After selection with hygromycin B, positive transformed calluses were obtained (Fig. 3-B). These calli were further cultured to obtain transgenic sweet potato seedlings (Fig. 4).
Fig. 3 Embryogenic callus co-culture with A. tumefaciens transformants (A) and selection of postively transformed callus with hygromycin (B)
Fig. 4 Seedlings of IbGBSSI-knockout lines after preliminary selections

(4) Verification of transgenic sweet potato plants

1) GUS detection

The GUS gene, is a commonly used reporter gene harboured in pCAMBIA1301s. Its expression product β-glucuronidase is a hydrolase that can catalyze the hydrolysis of many β-glucoside esters. It can decompose X-Gluc into blue substances, to observe the expression of foreign genes in transgenic plants and identify transgenic plants.
After the regenerated seedlings grew leaves, GUS staining was performed on these transgenic plants. Two successfully transformed sweet potato lines were preliminarily screened and designated as 23216004 and 23216005, whose leaves turned green (Fig. 5).
Fig. 5 Results of GUS staining

2) PCR detection

Genome DNA of these two transgenic lines was extracted from leaves of these two regenerated seedlings. PCR detection on the genomes was performed by using two pairs of primers which were designed based on the Cas9 protein gene and the hygromycin resistance gene (HygR), respectively. As the result, the transgenic lines 23216004 and 23216005 were further validated (Fig. 6).
The sequences of the two pairs of primers were as bellow:
  • CAS9-F: 5’-atggactataaggaccacgacgg-3’; CAS9-R: 5’-ttgtcgcctcccagctgagacag-3’
  • HygR-F: 5’-Atgaaaaagcctgaactcac-3’; HygR-R: 5’-ctatttctttgccctcggac-3’

Subsequently, these two transgenic lines and the wild-type B23 were planted with the method of cuttage in an experimental greenhouse to harvest starch-rich tubers.
Fig. 6 Detection results of PCRs for gene HygR and gene Cas9.

3) Determination of the expression level of IbGBSSI in root tubers

Two months after transplantation, the tubers of the transgenic lines were harvested. It showed that the number and size of the root tubers were largely consistent with the wild type (Fig. 7).
Fig. 7 Phenotypes of the IbGBSSI-knockout lines planted in greenhouse
RNA was extracted from the fresh root tubers and cDNA was obtained through reverse transcription later. Then, the relative expression level of the gene IbGBSSI was determined with the method of Quantitative Real-time PCR with the root-tuber cDNA as templates. The result showed that the relative expression level of IbGBSSI in root tubers of the transgenic lines (0.1063 and 0.2407) was much lower than that of the wild type (1.0000) (Fig. 8). It revealed that the knock-out of IbGBSSI in the pathway of starch synthesis was successful.
Fig. 8 Q-PCR result of the relative expression level of IbGBSSI in root tubers

(5) Starch analysis of transgenic sweet potato root tubers

Freshly harvested sweet potato tubers were cleaned, peeled, and sliced into small pieces. Starch was extracted from these pieces for qualitative and quantitative detection afterwards.

1) Qualitative detection of the starch components

When exposed to iodine, Amylose appears blue, while amylopectin appears reddish brown or purple red. Therefore, the component qualitative detection of the total starch from the transgenic lines was performed. As the result, the total starch of the transgenic lines appears reddish brown while that appears blue of the wild type (Fig. 9). It indicated that the total starch of the transgenic lines was composed mainly of amylopectin.
Fig. 9 Component detection of total starch by iodine staining