Part:BBa_K2201061:Design

GMPS iso2

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 24
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design

RNA Extraction

Croton tiglium samples were kindly provided by the botanical garden of the Phillips University in Marburg ("https://www.uni-marburg.de/botgart"). Total RNA was extracted from frozen tissue samples of young leaves, stem, inflorescence, seeds and roots. Mortar and pestle were used to grind the material in liquid nitrogen. The Spectrum Plant Total RNA Kit (http://www.sigmaaldrich.com/catalog/product/sigma/strn50?lang=de&region=DE&gclid=EAIaIQobChMI7ff3iqCe1wIVHsayCh3sww4YEAAYAiAAEgKfXvD_BwE) was used for the RNA extracting according to the suppliers’ instructions. mRNAs were enriched based on their polyA tail via oligo-dT beads.DropSense16 (trinean) (http://www.selectscience.net/products/dropsense16---micro-volume-spectrophotometer/?prodID=171883) was used for quality control.

Library Preparation

Enriched mRNA samples from different tissues were used for the construction of a normalized library (http://www.vertis-biotech.com/Home). In parallel, tissue specific samples were submitted to fragmentation prior to reverse transcription into cDNA via ProtoScript II (https://www.neb.com/products/m0368-protoscript-ii-reverse-transcriptase#Product%20Information)based on suppliers’ recommendations. The Illumina TruSeq Stranded mRNA Sample Preparation Guide (https://support.illumina.com/downloads/truseq_stranded_mrna_sample_preparation_guide_15031047.html) was used for the generation of five tissue specific libraries with an average insert size of 400 bp. Those libraries represent young leaves, stem, inflorescence, seed, and root.

Sequencing

Sequencing of the normalized library was performed on two lanes of an Illumina HiSeq1500 (https://support.illumina.com/sequencing/sequencing_instruments/hiseq_1500.html) generating about 47.4 million 2x250 nt paired-end reads. Sequencing of the tissue specific libraries was performed on a HiSeq1500 generating between 20 and 44 million 2x75 nt paired-end reads per tissue-specific library (Table 1).

After we had identified all interesting sequences from the purine pathway, we had to extract them from the cDNA of the tissue samples. Thus, we designed primers for the candidate sequence of isoform 2 of the GMPS that was identified in the transcriptome assembly of C. tiglium. In parallel to the cloning of native sequences, we ordered codon-optimized coding sequences for all candidate genes. The codon optimized sequence can be found within this part.

Protein Purification

A modification (https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--ImpactProtocol.pdf) of the NEB Impact® Kit https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale6901.pdf) was used for the purification of heterologous expression in E. coli. The protein purification via the Impact system works with the usage of an intein tag. Impact is short for “Intein Mediated Purification with Affinity Chitin-binding Tag”. In short, the coding sequence of the candidate gene is linked with an intein tag that enables the protein’s purification. As a vector, we used pTXB1 that is responsible for a c-terminal fusion of the intein tag. The expression strain ER2566 with the plasmid pRARE-2 was used. This vector is used to compensate for a bad codon usage as it encodes some rare tRNAs.

Concentrations of purified proteins were estimated based on a modification (https://static.igem.org/mediawiki/2017/6/6f/T--Bielefeld-CeBiTec--RotiNanoquant.pdf) of the Bradford Assay (Bradford, M., 1976) Roti®-Nanoquant (https://www.carlroth.com/downloads/ba/de/K/BA_K880_DE.pdf). Depending on the replicate, we reached concentrations from 1.46 up to 4.18 grams per liter (Table 2).

Source

n/a