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Team:Mingdao/bio-brick.html
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Biobrick Map- Click link to go to biobrick description
Part Registry Name Attribute Part (E-X-Part-S-P) Part Length Gene Gene Length
pSB1C3-Plac-NB (Part: BBa_K1256001)
BENEFIT: cloning genes with NheI & BamHI sites, easily exchange RFP gene
BIOBRICK LIGATION:
- “X-P” ligated to “S-P” ( X, S are compatible): standard part
- “E-N” ligated to “E-X” (N, X are compatible): to exchange front part (Fig 1. A)
- “N-P” ligated to “S-P” (N, S are compatible): to exchange back part (Fig 1. B)
Fig 1. BioBrick “part in part” (image below)
(A)(B)
pSB1C3-Plac-NB-Cm-MN (BBa_K1256002)
BENEFIT: cloning genes with MfeI & NsiI sites under CmR cassette
BIOBRICK LIGATION:
- “M-Ns” ligated to “E-P” ( M, E are compatible; Ns, P are compatible)
pSB1C3-Plac-SS-NB (Part: BBa_K1256003)
Secretion signal (SS): OmpA SS from E. coli
BENEFIT: cloning genes with NheI & BamHI sites, easily exchange RFP gene
BIOBRICK LIGATION:
- “X-P” ligated to “S-P” or “N-P” ligated to “E-X” (X, S, N are compatible)
pSB1C3-Plac-SS-Cecropin-MprF (BBa_K1256004)
MprF of Bacillus subtilis (DB2)added a RBS (BBa_B0034)was amplified by PCR and cloned into the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) using NheI and BamHI sites. Next, the cds of cecropin was synthesized on the primers and subjeted to Gibson Assembly to make this plasmid. The amino acid sequences of cecropin was obtained from Sigma-Aldrich, and the nucleotide sequence was optimized for engineering Escherichia coli using the codon optimization tool provided by Integrated DNA Technologies (IDT).
pSB1C3-Plac-SS-Magainin-MprF (BBa_K1256005)
MprF of Bacillus subtilis (DB2)added a RBS (BBa_B0034)was amplified by PCR and cloned into the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) using NheI and BamHI sites. Next, the cds of magainin was synthesized on the primers and subjeted to Gibson Assembly to make this plasmid. The amino acid sequences of magainin was obtained from Sigma-Aldrich, and the nucleotide sequence was optimized for engineering Escherichia coli using the codon optimization tool provided by Integrated DNA Technologies (IDT).
Colony PCR was performed to check the plasmid (Right Photo). The cecropin and magainin sequences were further confirmed by sequencing.
pSB1C3-Plac-SS-Protease (BBa_K1256006)
The protease coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and BamHI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003). The cds can be easily exchanged with NheI and BamHI. However, PstI site can no longer be used in this plasmid due to three more sites on the gene coding region.
pSB1C3-Plac-SS-Lipase (BBa_K1256007)
The lipase coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and NsiI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) with NheI and PstI, leaving a scar of NsiI/PsiI in the end.
pSB1C3-Plac-SS-DNase (BBa_K1256008)
The DNase coding sequence (cds) of Stenotrophomonas maltophilia D457 was obtained from KEGG Genes Database. The gene was cut by NheI and BamHI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003). The cds can be easily exchanged with NheI and BamHI. However, PstI site can no longer be used in this plasmid due to two more sites on the gene coding region.
pSB1C3-Plac-SS-RNase (BBa_K1256009)
The RNase coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and BglII and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) cut by NheI and BamHI. PstI site can no longer be used in this plasmid due to one more site on the gene coding region.
pSB1C3-Plac-SS-Chitinase (BBa_K1256010)
The chitinase coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and NsiI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) with NheI and PstI, leaving a scar of NsiI/PsiI in the end.
pSB1C3-Plac-SS-Glucanase (BBa_K1256011)
The glucanase coding sequence (cds) of Bacillus subtilis 168 was obtained from KEGG Genes Database. The gene was cut by NheI and BamHI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003). The cds can be easily exchanged with other BioBrick parts with NheI/PstI and SpeI/PstI, or be considered as a standard BioBrick parts (E-X-S-P).
Genes of decomposing enzymes were cloned either from Stenotrophomonas maltophilia or Bacillus subtilis. Figure 1 showed the genes were amplified by PCR.
Figure 1. PCR to amplify the genes of decomposing enzymes
(A)(B)
pSB1C3-Plac-RFP-Cm-LacI (BBa_K1256012)
RBS, LacI coding region and terminators (Part:BBa_Q04121) were amplified by PCR and cut by EcoRI and PstI. The PCR-amplified and enzyme-cut product was introduced to the novel designed plasmid backbone of pSB1C3-Plac-NB-Cm-MN ( Part:BBa_K1256002) with MfeI and NsiI. LacI gene is driven by Chloramphenicol promoter. RFP coding region can be exchanged using NheI and BamHI.
Figure 1 showed the result of colony PCR (A) and the RFG gene expression induced by IPTG (B).
(A)(B)
pSB1C3-Plac-ccdB-Cm-LacI (BBa_K1256013)
ccdB coding region was amplified by PCR from BBa_K145151 and exchanged with RFP on the plasmid of BBa_K1256012.
pSB1C3-Pred-ccdB-light sensor (BBa_K1256014)
Gene expression of ccdB (BBa_K145151), a lethal gene, is regulated by the light. The light-regulated system is composed of light sensor (BBa_K1017101) and light-regulated promoter (BBa_K1017301) designed and constructed by NCTU-Formosa in 2013. Light sensor was first amplified by PCR, cut by EcoRI and PstI, and ligated to pSB1C3-Plac-NB-Cm-MN cut by MfeI and NsiI. Then, light-regulated promoter was amplified by PCR, cut by EcoRI and NheI, and ligated to the resulting vector cut by EcoRI and NheI, which maintains the standard BioBrick assembly rule as well as the flexibility of exchanging RFP cds with NheI and BamHI. The ccdB was amplified by PCR using the forward primer with RBS and assembled to the vector with NheI and BamHI.
