Composite

Part:BBa_K4103012

Designed by: Almiro Pires da Silva Neto   Group: iGEM21_Open_Science_Global   (2021-10-12)


TEV-GFP-10xHis_R5 C-Tag

Composite part consisting of fluorescent marker conjugated with secretion tag.

Codon Optimized for Bacillus Subtilis in starvation environments.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 116
  • 1000
    COMPATIBLE WITH RFC[1000]


pHT43 is E. coli / B. subtilis shuttle vector. provides ampicillin resistance to E.coli and chloramphenicol resistance to B. subtilis.

https://www.lifescience-market.com/plasmid-c-94/pht43-p-63793.html Removed 2x BtgzI, 2x BbsI, and 1x BsmbI sites in the CDS region Maintained the BsmbI site in the promoter. Same with KpnI.

ccdB dropout derived from the p_Open_V3 FreeGenes Open Plasmids [BBF10K_003498] - Amino Acid codon 73 (V) changed from GTC -> GTG to remove a BsaI site.

Took the following design (see below for all info) and replaced the functional region from spisPink to ccdB (see above for source)

Documentation of AF_spisPink_cassette reproduced below (NOTE: not all of this may apply to our modified design)

Purpose This component contain the spisPink negative marker cassette to assemble the OE destination vector. The cassette is the same as Moebius Assembly but we change the promoter to J23119 (the stronger constitutive promoter of the series) to compensate for the lower copy number of pCO compared to pSB1K3 (used in Mobious)

OE acceptor schema: It drives constitutive spisPink chromoprotein expression as a negative maker reporter as in https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189892#sec011. It doesn't need any kind of supplementation to work. The constitutive promoter is the strongest of the the J23 series of promoters https://parts.igem.org/Part:BBa_J23119

SpisPink absorbance peak is 560nm giving a pink color visible to the naked eye in less than 24h, even with low copy number vectors https://journals.plos.org/plosone/article? --> after cloning white colonies are positive: they have lost the spisPink gene in place of your construct of choice

Assembly is performed with BsaI golden gate reaction beetween A and F sites. The whole insert is surrounded by SapI sequences, UNSes and SEVA cargo restriction enzymes (PacI and SpeI) and BbsI.

SapI: for uLoop Assembly UNS1/UNSX : To perform sequencing with standardized primers and to perform linear DNA gene expression. PacI + SpeI: To move the assembly as "SEVA cargo" to a SEVA vector. BbsI: To perform CIDAR lvl2 assembly.

[edit]
Categories
Parameters
None