Team:Saarland/4 step

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<h1>4. Build an army of clone warriors</h1>
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<h3>Design and synthesis of hyaluronic acid coding <i>has</i>2 gene</h3>
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<h2> More information is coming soon! </h2>
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The initial step of our project was the artificial synthesis of the <i>has</i>2 gene sequence coding for the hyaluronic acid synthase of the naked mole rat. The sequence was taken from the freely accessible genome database “UCSC genome browser”, whereby the sequence data was gained by shot-gun-sequencing (Kim <i>et al</i>., 2011). The gene sequence was adapted to the altered codon usage in <i>Bacillus megaterium</i> with JCat (Grote <i>et al</i>., 2005). Additionally the open reading frame (ORF) of the has2 gene was modified so that prokaryotic ribosomal binding site (RBS), rho-independent transcription termination as well as restriction enzymes that are also part of the multiple cloning site (MCS) of the target plasmid pSMF2.1 were excluded. For the upcoming cloning process of the <i>has</i>2 gene the nucleotide sequence was terminally modified with the restriction sites <i>Mlu</i>Ⅰ (5`end) und <i>Sac</i>Ⅰ(3`end). Moreover a </i>B. megaterium</i> specific RBS was added downstream the <i>Mlu</i>Ⅰrestriction site for efficient protein expression. We paid attention to the fact that amino acid sequence did not change.
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Subsequently the 1693 bp long modified gene sequence <i>has</i>2 (fig. 1) was handed over to Eurofins who performed the gene synthesis and the cloning into the standard delivery plasmid pexK4 plasmid (Eurofins).<br>
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<!-- [[File:|thumb|center|400px|<b>Figure 1: Modified gene sequence of the hyaluronan synthase <i>has</i>2 of the naked mole rat (1693 bp).</b> The start codon and stop codon of the has2-sequence are marked (red). At the 5`end the restriction site <i>Mlu</i>Ⅰ(green) and the RBS (yellow) is annexed. At the 3`end the recognition site of the <i>Sac</i>Ⅰ enzyme is added. (Modified from UCSC Genome Browser)]] -->
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<h3>Genomic amplification of genes that may optimise the hyaluronic acid production</h3>
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The hyaluronan synthase catalyses the synthesis of hyaluronic acid by consumption of the endogenous synthesised precursor molecules UDP-GlcUA and UDP-GlcNAc. The biochemical pathways for their production were already identified in streptococci of group A and C. Hence it was possible to identify genes that could have a beneficial effect on the concentration of these precursor molecules for high yield hyaluronic acid production in <i>B. megaterium</i>
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<!--[Hyperlink to Pathway engineering Teil 2].-->
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Eventually eight relevant pathway genes, including <i>UDP-Glc</i>DH, <i>glk</i>, <i>pgm</i>B, <i>gta</i>B, <i>pgi, glm</i>S,<i> glm</i>M and <i>gca</i>D, were chosen for the approach of pathway engineering. The sequences of these genes originate from MegaBac v9 database and were used for primer design and amplification of genomic DNA of <i>B. megaterium.</i> Restriction sites not only at the 5` end but also at the 3` end were added for subsequent cloning into the pSB1C3 plasmid (table 1). <br>
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{|
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|+<b>Table 1:</b> Overview of the BioBrick length and BioBrick restriction sites
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!Gene Name
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!Gene Size (bp)
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!5` Restriction Site
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!3` Restriction Site
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|<i>UDP-Glc</i>DH
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|1382
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|<i>Sac</i>Ⅰ
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|<i>Sph</i>Ⅰ
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|-
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|<i>glk</i>
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|969
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|<i>EcoR</i>Ⅰ
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|<i>Pst</i>Ⅰ
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|-
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|<i>pgm</i>B
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|707
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|<i>EcoR</i>Ⅰ
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|<i>Spe</i>Ⅰ
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|-
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|<i>gta</i>B
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|888
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|<i>EcoR</i>Ⅰ
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|<i>Pst</i>Ⅰ
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|-
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|<i>pgi</i>
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|1350
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|<i>Xba</i>Ⅰ
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|<i>Pst</i>Ⅰ
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|-
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|<i>glm</i>S
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|1800
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|<i>Xba</i>Ⅰ
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|<i>Pst</i>Ⅰ
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|-
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|<i>glm</i>M
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|1346
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|<i>Eco</i>RⅠ
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|<i>Pst</i>Ⅰ
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|-
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|<i>gca</i>D
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|1380
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|<i>Eco</i>RⅠ
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|<i>Pst</i>Ⅰ
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|-
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|}
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Revision as of 11:22, 15 October 2014


4. Build an army of clone warriors


Design and synthesis of hyaluronic acid coding has2 gene

The initial step of our project was the artificial synthesis of the has2 gene sequence coding for the hyaluronic acid synthase of the naked mole rat. The sequence was taken from the freely accessible genome database “UCSC genome browser”, whereby the sequence data was gained by shot-gun-sequencing (Kim et al., 2011). The gene sequence was adapted to the altered codon usage in Bacillus megaterium with JCat (Grote et al., 2005). Additionally the open reading frame (ORF) of the has2 gene was modified so that prokaryotic ribosomal binding site (RBS), rho-independent transcription termination as well as restriction enzymes that are also part of the multiple cloning site (MCS) of the target plasmid pSMF2.1 were excluded. For the upcoming cloning process of the has2 gene the nucleotide sequence was terminally modified with the restriction sites MluⅠ (5`end) und SacⅠ(3`end). Moreover a </i>B. megaterium</i> specific RBS was added downstream the MluⅠrestriction site for efficient protein expression. We paid attention to the fact that amino acid sequence did not change. Subsequently the 1693 bp long modified gene sequence has2 (fig. 1) was handed over to Eurofins who performed the gene synthesis and the cloning into the standard delivery plasmid pexK4 plasmid (Eurofins).



Genomic amplification of genes that may optimise the hyaluronic acid production

The hyaluronan synthase catalyses the synthesis of hyaluronic acid by consumption of the endogenous synthesised precursor molecules UDP-GlcUA and UDP-GlcNAc. The biochemical pathways for their production were already identified in streptococci of group A and C. Hence it was possible to identify genes that could have a beneficial effect on the concentration of these precursor molecules for high yield hyaluronic acid production in B. megaterium 

Eventually eight relevant pathway genes, including UDP-GlcDH, glk, pgmB, gtaB, pgi, glmS, glmM and gcaD, were chosen for the approach of pathway engineering. The sequences of these genes originate from MegaBac v9 database and were used for primer design and amplification of genomic DNA of B. megaterium. Restriction sites not only at the 5` end but also at the 3` end were added for subsequent cloning into the pSB1C3 plasmid (table 1).


Table 1: Overview of the BioBrick length and BioBrick restriction sites
Gene Name Gene Size (bp) 5` Restriction Site 3` Restriction Site
UDP-GlcDH 1382 SacⅠ SphⅠ
glk 969 EcoRⅠ PstⅠ
pgmB 707 EcoRⅠ SpeⅠ
gtaB 888 EcoRⅠ PstⅠ
pgi 1350 XbaⅠ PstⅠ
glmS 1800 XbaⅠ PstⅠ
glmM 1346 EcoRⅠ PstⅠ
gcaD 1380 EcoRⅠ PstⅠ




















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