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PMID:39007160 experiment design (#235)
* experiment desing from PMID:39007160 (Ziqiang et al. 2024) * update html form
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‎batch_cloning/__init__.py‎

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‎batch_cloning/ziqiang_et_al2024.html‎

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<!DOCTYPE html>
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<html>
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<head>
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<title>Enter DNA Sequences</title>
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<style>
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body {
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font-family: 'Segoe UI', Tahoma, Geneva, Verdana, sans-serif;
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max-width: 800px;
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margin: 0 auto;
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padding: 20px;
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text-align: center;
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}
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textarea {
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width: 400px;
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height: 300px;
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margin: 20px auto;
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display: block;
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font-family: monospace;
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}
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.error {
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color: red;
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margin: 10px auto;
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}
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input[type="submit"] {
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background-color: #4CAF50;
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border: none;
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color: white;
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padding: 15px 32px;
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text-align: center;
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text-decoration: none;
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display: inline-block;
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font-size: 1.4em;
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margin: 4px 2px;
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cursor: pointer;
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border-radius: 4px;
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transition: background-color 0.3s;
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}
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input[type="submit"]:hover {
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background-color: #45a049;
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}
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</style>
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</head>
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<body>
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<h1>Versatile Cloning Strategy for Efficient Multigene Editing in Arabidopsis</h1>
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<p>Ziqiang P. Li, Jennifer Huard, Emmanuelle M. Bayer and Valérie Wattelet-Boyer</p>
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<p><a href="https://doi.org/10.21769/BioProtoc.5029">doi:10.21769/BioProtoc.5029</a></p>
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<p>Enter protospacer sequences (20 bases each) separated by line breaks.</p>
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<p>Below is the example protospacer sequences from the paper.</p>
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<form id="sequenceForm">
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<textarea id="sequences" name="sequences">
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GCTGGCTAACCGTGAGGGGA
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CCGTGTACTGTAGTTACAGT
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TGTGGTTCCCCGGCCGTCTT
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ATACTCTAGTCCTCAACGCC</textarea>
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<br>
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<div id="error" class="error">
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</div>
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<input type="submit" value="Submit">
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</form>
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<script>
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document.getElementById('sequenceForm').addEventListener('submit', async function (e) {
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e.preventDefault();
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const sequences = document.getElementById('sequences').value;
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const lines = sequences.split('\n').filter(line => line.trim().length > 0);
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const dnaRegex = /^[ACGTacgt]{20}$/;
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const valid = lines.every(line => dnaRegex.test(line.trim()));
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const errorDiv = document.getElementById('error');
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errorDiv.textContent = valid ? '' : 'Each sequence must be exactly 20 bases long and contain only A, C, G, or T';
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if (valid) {
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// Submit the form data via fetch
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try {
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const response = await fetch(window.location.href, {
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method: 'POST',
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headers: {
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'Content-Type': 'application/json',
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},
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body: JSON.stringify(lines)
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});
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if (!response.ok) {
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if (response.status === 400) {
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const error = await response.json();
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errorDiv.textContent = error.detail;
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} else {
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throw new Error(`HTTP error! status: ${response.status}`);
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}
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}
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const data = await response.json();
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// Create a blob with the JSON data
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const blob = new Blob([JSON.stringify(data, null, 2)], { type: 'application/json' });
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// Create a temporary link element
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const downloadLink = document.createElement('a');
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downloadLink.href = URL.createObjectURL(blob);
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downloadLink.download = 'cloning_strategy.json';
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// Trigger the download
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document.body.appendChild(downloadLink);
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downloadLink.click();
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// Clean up
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document.body.removeChild(downloadLink);
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URL.revokeObjectURL(downloadLink.href);
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} catch (error) {
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console.error('Error:', error);
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}
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}
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});
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</script>
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</body>
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</html>

‎batch_cloning/ziqiang_et_al2024.json‎

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‎batch_cloning/ziqiang_et_al2024.py‎

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from Bio.Seq import reverse_complement
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from fastapi import APIRouter
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from fastapi.responses import FileResponse
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from pathlib import Path
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router = APIRouter()
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def validate_protospacers(protospacers):
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"""
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Ensure that the protospacers are compatible for the golden gate assembly, by checking that the central 4
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bps are different in all of them, and also different from the other assembly joints.
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"""
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forbidden_joints = ['AACA', 'GCTT']
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for i, ps in enumerate(protospacers):
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# Must be only ACGT
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if not set(ps).issubset({'A', 'C', 'G', 'T'}):
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raise ValueError(f"Protospacer {i} {ps} contains invalid bases")
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if len(ps) != 20:
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raise ValueError(f"Protospacer {i} {ps} is not 20 bp long")
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if ps[8:12] in forbidden_joints:
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raise ValueError(
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f"Protospacer {i} has a forbidden joint {ps[8:12]}, which is used for the constant parts of the assembly"
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)
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# Find any other protospacers with the same joint sequence
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same_joint_indexes = [
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j + 1 for j, other_ps in enumerate(protospacers) if i != j and ps[8:12] == other_ps[8:12]
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]
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if same_joint_indexes:
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raise ValueError(
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f"Protospacer {i + 1} has the same joint as protospacers {', '.join(map(str, same_joint_indexes))}"
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)
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def design_primers(protospacers):
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fwd_prefix = 'taggtctcc'
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rvs_prefix = 'atggtctca'
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fwd_suffix = 'gttttagagctagaa'
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rvs_suffix = 'tgcaccagccgggaa'
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primers = list()
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for protospacer in protospacers:
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primers.append(rvs_prefix + reverse_complement(protospacer[:12]) + rvs_suffix)
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primers.append(fwd_prefix + protospacer[8:] + fwd_suffix)
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return primers
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@router.get('/batch_cloning/ziqiang_et_al2024')
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def ziqiang_et_al2024():
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return FileResponse(Path(__file__).parent / 'ziqiang_et_al2024.html')

‎main.py‎

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import io
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from Bio import BiopythonParserWarning
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from gateway import gateway_overlap, find_gateway_sites, annotate_gateway_sites
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from batch_cloning.ziqiang_et_al2024 import (
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router as ziqiang_et_al2024_router,
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validate_protospacers,
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design_primers as design_primers_ziqiang_et_al2024,
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)
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import json
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# ENV variables ========================================
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RECORD_STUBS = os.environ['RECORD_STUBS'] == '1' if 'RECORD_STUBS' in os.environ else False
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raise HTTPException(404)
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@router.post('/batch_cloning/ziqiang_et_al2024', response_model=BaseCloningStrategy)
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async def ziqiang_et_al2024_post(protospacers: list[str]):
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try:
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validate_protospacers(protospacers)
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except ValueError as e:
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raise HTTPException(400, str(e))
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primers = design_primers_ziqiang_et_al2024(protospacers)
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with open('batch_cloning/ziqiang_et_al2024.json', 'r') as f:
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template = BaseCloningStrategy.model_validate(json.load(f))
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max_primer_id = max([primer.id for primer in template.primers], default=0)
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for i, primer in enumerate(primers):
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max_primer_id += 1
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orientation = 'rvs' if i % 2 == 0 else 'fwd'
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template.primers.append(
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PrimerModel(id=max_primer_id, name=f"protospacer_{i // 2 + 1}_{orientation}", sequence=primer)
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)
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primer_ids_for_pcrs = [3, *[p.id for p in template.primers[-len(primers) :]], 12]
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next_node_id = max([s.id for s in template.sequences] + [s.id for s in template.sources]) + 1
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template_sequence = next(s for s in template.sequences if s.id == 18)
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for i, (fwd_primer_id, rvs_primer_id) in enumerate(zip(primer_ids_for_pcrs[::2], primer_ids_for_pcrs[1::2])):
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pcr_source = PCRSource(id=next_node_id, output_name=f"pcr_protospacer_{i + 1}")
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fwd_primer = next(p for p in template.primers if p.id == fwd_primer_id)
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rvs_primer = next(p for p in template.primers if p.id == rvs_primer_id)
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next_node_id += 1
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resp = await pcr(pcr_source, [template_sequence], [fwd_primer, rvs_primer], 14, 0)
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pcr_product: TextFileSequence = TextFileSequence.model_validate(resp['sequences'][0])
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pcr_product.id = next_node_id
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pcr_source: PCRSource = PCRSource.model_validate(resp['sources'][0])
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pcr_source.output = next_node_id
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template.sequences.append(pcr_product)
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template.sources.append(pcr_source)
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next_node_id += 1
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# Find all PCR products
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# (we use type instead of isinstance because the BaseCloningStrategy does not
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# have the newer source models with extra methods)
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pcr_product_ids = [s.output for s in template.sources if s.type == 'PCRSource']
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# Make all input of a Golden gate assembly
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golden_gate_source = RestrictionAndLigationSource(
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id=next_node_id, output_name='golden_gate_assembly', restriction_enzymes=['BsaI'], input=pcr_product_ids
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)
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next_node_id += 1
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# Make them
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input_sequences = [next(s for s in template.sequences if s.id == p) for p in pcr_product_ids]
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resp = await restriction_and_ligation(golden_gate_source, input_sequences, False, False)
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golden_gate_product: TextFileSequence = TextFileSequence.model_validate(resp['sequences'][0])
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golden_gate_product.id = next_node_id
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golden_gate_source: RestrictionAndLigationSource = RestrictionAndLigationSource.model_validate(resp['sources'][0])
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golden_gate_source.output = next_node_id
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next_node_id += 1
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template.sequences.append(golden_gate_product)
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template.sources.append(golden_gate_source)
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bp_target = next(s for s in template.sequences if s.id == 12)
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gateway_source = GatewaySource(id=next_node_id, output_name='entry_clone', reaction_type='BP', greedy=False)
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next_node_id += 1
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resp = await gateway(gateway_source, [golden_gate_product, bp_target], circular_only=True, only_multi_site=True)
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gateway_product: TextFileSequence = TextFileSequence.model_validate(resp['sequences'][0])
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gateway_product.id = next_node_id
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gateway_source: GatewaySource = GatewaySource.model_validate(resp['sources'][0])
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gateway_source.output = next_node_id
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next_node_id += 1
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template.sequences.append(gateway_product)
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template.sources.append(gateway_source)
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# Now we want to do a Gateway with everything, so we need to find all sequences that are not input of anything
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all_input_ids = sum([s.input for s in template.sources], [])
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sequences_to_clone = [s for s in template.sequences if s.id not in all_input_ids]
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gateway_source = GatewaySource(id=next_node_id, output_name='expression_clone', reaction_type='LR', greedy=False)
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next_node_id += 1
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resp = await gateway(gateway_source, sequences_to_clone, circular_only=True, only_multi_site=True)
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index_of_product = next(i for i, s in enumerate(resp['sequences']) if '/label="Cas9"' in s.file_content)
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expression_clone: TextFileSequence = TextFileSequence.model_validate(resp['sequences'][index_of_product])
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expression_clone.id = next_node_id
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gateway_source: GatewaySource = GatewaySource.model_validate(resp['sources'][index_of_product])
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gateway_source.output = next_node_id
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next_node_id += 1
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template.sequences.append(expression_clone)
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template.sources.append(gateway_source)
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return template
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app.include_router(router)
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app.include_router(ziqiang_et_al2024_router)

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