Introduction

For our project, we decided to submit seven BioBricks to the iGEM Registry in the pSB1C3 plasmid backbone. Our submitted parts can be divided into two separate collections (Named as Collection One and Collection Two). Collection One is associated with Engineered biofilm subunit CsgA, while Collection Two is relevant to construction of hydrogenase gene clusters.

All of the constructs are protein-coding parts have been confirmed with gene sequences, demonstrated with expected functionalities, and thus it would be easy to handle for other users. Specifically, in order to make hydrogenase expression more productive, we optimized the gene sequences of 5 hydrogenases parts with the help of OptimumGene™ (GenScript). We changed the codon usage bias in E. coli by upgrading the CAI (Codon Adaptation Index) from a low level (around 0.30) to 0.97.

Characterizations of the parts are included on the webpage of the parts section, but for more information about the submitted parts in real use, please go to Biofilm or Hydrogen page.

Basic Parts

Name	Type	Description	Designer	Length
BBa_K2132001	Coding	CsgASpyCatcherHisTag	Shijie Gu	867
BBa_K2132003	Coding	mCherry-SpyTag	Shijie Gu	813
BBa_K2132004	Coding	HydA with SpyCatcher, Histag and TEV site	Yifan Chen	2166
BBa_K2132005	Coding	HydA with SpyTag, Histag and TEV site	Yifan Chen	1857
BBa_K2132006	Coding	HydE with Histag and TEV site	Yifan Chen	1098
BBa_K2132007	Coding	HydF with Histag and TEV site	Yifan Chen	1281
BBa_K2132008	Coding	HydG with Histag and TEV site	Yifan Chen	1464

Part Collection

1.Parts Collection One (Engineered Biofilm Subunit CsgA with SpyCatcher and HisTag)

a) In order to realize covalent link of two proteins, we make use of SpyTag and SpyCatcher system (see our Linkage System on Biofilm Page). The SpyTag is fused to HydA of the hydrogenase system, thus a SpyCatcher on CsgA would enable Spy-tagged Hydrogenase to be covalently attached to CsgA In the meantime, to endow biofilm CsgA protein with additional functionalities (such as specific binding to QDs or Nanorods in our case), we append one or two HisTag at the N- or/and C-terminus of the CsgA-spycatcher protein. This lead to two new constructs, HisTag-CsgA-SpyCatcher-HisTag (BBa_K2132001) and HisTag-CsgA-SpyCatcher (the former being sequence confirmed and submitted).

➤ CsgASpyCatcherHisTag (E. coli) - BBa_K2132001

➤ mCherry-SpyTag (E. coli) - BBa_K2132003

b) To determine if expression of the two proteins is successful, we constructed mCherry-SpyTag to test the activity of the SpyCatcher on the CsgA. mCherry-SpyTag is thus submitted as BBa_K2132003, sequence confirmed.

2.Parts Collection Two (Hydrogenase gene clusters)

We optimized [FeFe] Hydrogenases originally from the bacterium Clostridium acetobutylicum (optimized coding sequence: hydA, BBa_K2132004 & BBa_K2132005) to accept electrons and therefor enable catalytic production of hydrogen in our project. Synthesis of heterologous [FeFe] hydrogenase in E. coli requires co-expression of HydE (optimized coding sequence: hydE, BBa_K2132006), HydF (optimized coding sequence: hydF, BBa_K2132007), and HydG (optimized coding sequence: hydG, BBa_K2132008).

In this collection, we sub-cloned the coding sequence into the pSB1C3 individually, with two appended tags at the N-terminus (His-tag to faciliate purification and TEV site as cleavable site for Histag cutting off). We have confirmed that the presence of the two tags won’t disrupt expression and normal functionalites of HydA.

➤ HydA with SpyCatcher, Histag and TEV site (E. coli) - BBa_K2132004

➤ HydA with SpyTag, Histag and TEV site (E. coli) - BBa_K2132005

➤ HydE with Histag and TEV site (E. coli) - BBa_K2132006

➤ HydF with Histag and TEV site (E. coli) - BBa_K2132007

➤ HydG with Histag and TEV site (E. coli) - BBa_K2132008

★Optimization of this collection

The original sequences of hydrogenase were found in www.genome.jp. With the help of OptimumGene™, We used the following parameters to optimize our gene sequences without changing their amino acids sequence: Codon usage bias, GC content, CpG dinucleotides content, mRNA secondary structure, Cryptic splicing sites, Premature PolyA sites, Internal chi sites and ribosomal binding sites, Negative CpG islands, RNA instability motif (ARE), Repeat sequences (direct repeat, reverse repeat, and Dyad repeat).

Reference

King P W, Posewitz M C, Ghirardi M L, et al. Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System[J]. Journal of Bacteriology, 2006, 188(6):2163-72.

@@ Line 131: / Line 131: @@
 b) To determine if  expression of the two proteins is successful, we constructed mCherry-SpyTag to test the activity of the SpyCatcher on the CsgA. mCherry-SpyTag is thus submitted as <a href="http://parts.igem.org/Part:BBa_K2132003">BBa_K2132003</a>, sequence confirmed.
         <h4>2.Parts Collection Two (Hydrogenase gene clusters)</h4>
-We utilize [FeFe] Hydrogenases originally from the bacterium Clostridium acetobutylicum (coding sequence: hydA, <a href="http://parts.igem.org/Part:BBa_K2132004">BBa_K2132004</a> & <a href="http://parts.igem.org/Part:BBa_K2132005">BBa_K2132005</a>) to accept electrons and therefor enable catalytic production of hydrogen in our project. Synthesis of heterologous [FeFe] hydrogenase in <em>E. coli</em> requires co-expression of HydE (coding sequence: hydE, <a href="http://parts.igem.org/Part:BBa_K2132006">BBa_K2132006</a>), HydF (coding sequence: hydF, <a href="http://parts.igem.org/Part:BBa_K2132007">BBa_K2132007</a>), and HydG (coding sequence: hydG, <a href="http://parts.igem.org/Part:BBa_K2132008">BBa_K2132008</a>).
+We optimized [FeFe] Hydrogenases originally from the bacterium Clostridium acetobutylicum (optimized coding sequence: hydA, <a href="http://parts.igem.org/Part:BBa_K2132004">BBa_K2132004</a> & <a href="http://parts.igem.org/Part:BBa_K2132005">BBa_K2132005</a>) to accept electrons and therefor enable catalytic production of hydrogen in our project. Synthesis of heterologous [FeFe] hydrogenase in <em>E. coli</em> requires co-expression of HydE (optimized coding sequence: hydE, <a href="http://parts.igem.org/Part:BBa_K2132006">BBa_K2132006</a>), HydF (optimized coding sequence: hydF, <a href="http://parts.igem.org/Part:BBa_K2132007">BBa_K2132007</a>), and HydG (optimized coding sequence: hydG, <a href="http://parts.igem.org/Part:BBa_K2132008">BBa_K2132008</a>).
 <p></p>
 In this collection, we sub-cloned the coding sequence into the pSB1C3 individually, with two appended tags at the N-terminus (His-tag to faciliate purification and TEV site as cleavable site for Histag cutting off). We have confirmed that the presence of the two tags won’t disrupt expression and normal functionalites of HydA.

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