Plasmid producing corn NAAT1, DMAS, and TOM1
NAAT1: NCBI. (19 October, 2011). Zea mays ZmNAAT1 mRNA for putative nicotianamine aminotransferase, complete cds. http://www.ncbi.nlm.nih.gov/nuccore/166788521 DMAS: NCBI. (2014). deoxymugineic acid synthase1 [Zea mays]. http://www.ncbi.nlm.nih.gov/protein/162460852 TOM1: NCBI. (10 December, 2008). Zea mays clone 222622 protein transporter mRNA, complete cds. http://www.ncbi.nlm.nih.gov/nuccore/195621011
This plasmid codes for NAAT1 (BBa_K1422502) and DMAS (BBa_K1422503), two of the four enzymes used to produce corn phytosiderophores; TOM1 (BBa_K1422507), which transports phytosiderophores out of cells; and sfGFP, as a reporter. BBa_K1422505 goes along with this plasmid, producing the other two enzymes necessary for the production of corn phytosiderophores, and includes a killswitch.
Using a conditional promoter so that the modified Bacillus subtilis would only produce phytosiderophores in the presence of plants roots was considered, but it was decided that conditional promoters would not be reliable enough. A constitutive promoter of medium strength was chosen for the corn genes because a weak promoter might not have produced enough phytosiderophores to make a difference, and a strong promoter might have overtaxed the cell in the production of phytosiderophores as to be counterproductive. The reporter is placed after the corn genes because gene expression decreases linearly as the distance from the promoter increases, and expressing the corn genes is much more important than expressing the reporter. sfGFP was used as a reporter because it is easily observed, easily measured, and expressed in Bacillus subtilis. The RBS SpoVG was chosen for its relative strength in Bacillus subtilis. A relatively strong terminator with few base pairs was selected for its relative effectiveness and low number of base pairs. NAAT can be coded by naat-A, naat-B, or naat-1; naat1 from corn was selected because a previous experiment revealed that the rice naat1 gene encodes functional NAAT and is expressed in cells of Fe-deficient leaves, strongly suggesting that it is involved in phytosiderophores production and sufficient to produce NAAT on its own, without other naat genes.
modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pairs
Artificial terminator, estimated %T~>90% *8bp stem, 6nt loop *Bidirectional, estimated reverse %T~>90%
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 9 residues.
Promoter veg a constitutive promoter for B. subtilis
The Pveg promoter was suggested to us by Dr. Jan-Willem Veening of Newcastle University. This sequence supplied was compared to that of the DBTBS database#3 then a section containing the binding site synthesised by Geneart.
Pveg is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. Pveg contains binding sites for the ''B.sutbilis'' major sigma factor#1. Pveg in ''B.subtilis'' utilises two binding sites to cause high expression of genes#2, however our Pveg is lacking the upstream site to give a medium level of gene expression. It has been noted that the sporulation master regulatoion factor spoOA interacts with Pveg though it is not known how#3. The context with which we used the promoter Pveg is as a '''Polymerase Per Second''' (PoPS) generator.
The biobrick part was designed to include a single binding site for the ''B.subtilis major sigma factor. In addition the biobrick standard was applied to the promoter Pveg sequence.
SpoVG ribosome binding site (RBS) for B. subtilis
The sequence was taken from a previous research paper [1] and was constructed by Geneart.
Description: SpoVG is an endogenous ribosome binding site from B.subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B.subtilis. Previous research showed that the predicted binding energy of the 16s rRNA to the RBS is -19kcal 1
In order to ensure that the RBS is functional the actual ribosome binding site was maintained and the distance between the RBS and the start codon maintained. In order to conform to the biobrick standard the sequence flanking the RBS had to be changed but the distance between the promoter and RBS, and start codon and RBS was maintained.
Superfolder GFP was originally described by: Pedelacq et al (2006): "Engineering and characterization of a superfolder green fluorescent protein", Nature Biotech 24 (1) January 2006 This version was synthesised de novo (by Geneart).
This is the coding sequence of superfolder GFP (Pedelacq et al (2006): "Engineering and characterization of a superfolder green fluorescent protein", Nature Biotech 24 (1) January 2006). It carries the following amino acid changes with respect to mut3 GFP (E0040), the currently most commonly used GFP in the registry: S30R, Y39N, F64L, G65T, F99S, N105T, Y145F, M153T, V163A, I171V, A206V Its in-vivo properties are considerably improved with respect to mut3 - it develops fluorescence about 3fold faster than mut3 GFP and reaches 4fold higher absolute fluorescence levels. Fluorescenct colonies can be identified with the naked eye even without UV or blue light illumination (that is to say the amount of blue light in normal daylight or lablight is sufficient). Additionally it is more stable in vitro and refolds faster after in vitro denaturation with respect to mut3 GFP. Note: Superfolder GFP is available in constructs driven by the pBAD and T7 promoters: part numbers I746908 and I746909 respectively. Additionally 6-his tagged versions for protein purification exist: I746914 (pBAD driven) and I746915 (T7 driven).
Codon optimisation before de novo synthesis was carried out for both, E.coli and Bacillus subtilis.
Zea mays TOM1 (corn phytosiderophore transporter)
The original, unaltered gene sequence came from NCBI. NCBI. (10 December, 2008). Zea mays clone 222622 protein transporter mRNA, complete cds. http://www.ncbi.nlm.nih.gov/nuccore/195621011
Zea mays TOM1 is a transporter protein for corn phytosiderophores, allowing them to exit corn cells.
Codon optimized for Bacillus subtilis. The 60th, 283rd, 293rd, and 295th codons were altered from CTA to CTT.
Zea mays Nicotianamine aminotransferase (corn NAAT)
The original sequence was obtained from the NCBI website. NCBI. (19 October, 2011). Zea mays ZmNAAT1 mRNA for putative nicotianamine aminotransferase, complete cds. http://www.ncbi.nlm.nih.gov/nuccore/166788521
Nicotianamine aminotransferase (NAAT) catalases the conversion of nictotianamine to 3???deamino-3???-oxonicotianamine (a 3???-keto intermediate) and L-glutamate using 2-oxoglutarate. This is the third of four enzymes used to produce corn phytosiderophores and BBa_K1422500, BBa_K1422501, and BBa_K1422503 are also required to produce a phytosiderophore. Specifically, this is ZmNAAT1, although there are different forms among various species and types NAAT-A, NAAT-B, and NAAT1. Note: this sequence has been codon optimized for Bacillus subtilis.
Codon optimized for Bacillus subtilis by altering the 23rd, 109th, and 168th amino acid sequences from CTA to CTT.
Zea mays deoxymugineic acid synthase (corn DMAS)
The sequence for this was obtained from the NCBI website. NCBI. (2014). deoxymugineic acid synthase1 [Zea mays]. http://www.ncbi.nlm.nih.gov/protein/162460852
Deoxymugineic acid synthase (DMAS) reduces the 3"-carbon of 3???deamino-3???-oxonicotianamine (a 3???-keto intermediate) to produce 2???-Deoxymugineic Acid (DMA). This is the fourth of four enzymes used to produce corn phytosiderophores, and to produce a phytosiderophore would be used with BBa_K1422500, BBa_K1422501, and BBa_K1422502.
This sequence did not need to be altered to allow for Bacillus subtilis codon optimization.
Conversion of the iGEM parts registry to SBOL2.1