Difference between revisions of "Part:BBa K2703008"
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=='''Introduction'''==
 
=='''Introduction'''==
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For the silver medal criteria “Validated Part” we submit a basic part: the sequence of resistance marker to paromomycine that is an aminoglycoside antibiotic. The aphVIII gene from Streptomyces rimosus encode for an aminoglycoside 3’-phosphotransferase gene (aph). From the aphVIII gene sequence of the BioBrick BBa_K1884013 (iGEM16_SZU-China) XXX’s restriction site was removed ( G551C) by PCR point mutation.  
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For the silver medal criteria “Validated Part” we submit a basic part: the sequence of resistance marker to paromomycine that is an aminoglycoside antibiotic. The aphVIII gene from Streptomyces rimosus encode for an aminoglycoside 3’-phosphotransferase gene (aph). From the aphVIII gene sequence of the BioBrick BBa_K1884013 (iGEM16_SZU-China) BpiI estriction site were removed ( G551C) by PCR point mutation.  
 
We cloned aphVIII  by PCR amplification in B4-B5 position of the MoClo standard2 to use it in our retrotransposon design. Acceptor plasmid is the MoClo Universal pL0 acceptor plasmid pAGM91212. We sequenced again the sequence to be sure there is no unwanted mutations.  
 
We cloned aphVIII  by PCR amplification in B4-B5 position of the MoClo standard2 to use it in our retrotransposon design. Acceptor plasmid is the MoClo Universal pL0 acceptor plasmid pAGM91212. We sequenced again the sequence to be sure there is no unwanted mutations.  
  

Revision as of 17:11, 14 October 2018

paromycin resistance gene

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

For the silver medal criteria “Validated Part” we submit a basic part: the sequence of resistance marker to paromomycine that is an aminoglycoside antibiotic. The aphVIII gene from Streptomyces rimosus encode for an aminoglycoside 3’-phosphotransferase gene (aph). From the aphVIII gene sequence of the BioBrick BBa_K1884013 (iGEM16_SZU-China) BpiI estriction site were removed ( G551C) by PCR point mutation. We cloned aphVIII by PCR amplification in B4-B5 position of the MoClo standard2 to use it in our retrotransposon design. Acceptor plasmid is the MoClo Universal pL0 acceptor plasmid pAGM91212. We sequenced again the sequence to be sure there is no unwanted mutations.


1- Biological background

It is usually use in Chlamydomonas reinhardtii under the control of the HSP70A promoter and the 3’UTR of RBCS2 gene as terminator1. Here we report the domestication of the aphVIII gene in the C. reinhardtii MoClo Kit

2- Usage in iGEM projects

Our part is compatible with the PhytoBrick MoClo standard. This PhytoBrick was intended to be used as a rapporter gene in the CARGO to test the functionality of our synthetic retrotransposons for directed evolution in C. reinhardtii.

Sequencing

Characterization

We couldn’t directly use our Phytobrick for its characterization because we did not have the time to construct all the other parts needed for the final functional assembly. Instead we used a slightly different Phytobrick with the same aphVIII sequence but with different fusion site in 5’ and 3’.

Name F1 : B4 PART F2: B5
pL0-Paro AGGT aphVIII TTCG
Figure 1 : Schematic representation of the Phytobrick BBa_K2703008 and its fusion site for the MoClo assembly. It complies with the Common Genetic Syntax fusion sites.


This part was assembled into a functional Transcription Unit with. After transformation in C. reinhardtii D66 strain3 the functionality aphVIII gene sequence was tested by counting the number of colony resistant to paromomycine (15 ug/mL).
Name F1 Prom F2 5'UTR F3 Resistance F4 3'UTR F5
pCM-1 GGAG PAR TACT 5’UTRAR AATG Paromycin GCTT TRBCS2 CGCT
Figure 2: The 4 different devices made to test the functionality of the promoter PPSAD. The construction were made with the MoClo kit made for C.reinhardtii by P.Crozet et al 2018 1.
Figure 3: Graphic listing the 4 devices (pCM-1) transformed in C.reinhardtii D66 by electroporation with 100 ng of DNA. The selection was made on TAP agar media with paromomycin (15 g/mL) by plate. Data are mean  SD (N=3) and there were no colonies with a negative control .
The aphVIII gene sequence work as intented in once a transcription unit is assembled by GoldenGate cloning following the Chlamydomonas MoClo kit.

References

  1. Sizova, I., Fuhrmann, M. & Hegemann, P. A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277, 221–229 (2001).

    2. DOI: 10.1021/acssynbio.8b00251

  2. Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A modular cloning system for standardized assembly of multigene constructs. PLoS One 6, (2011).
  3. Crozet, P. et al. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth. Biol. (2018). doi:10.1021/acssynbio.8b00251