Basic Parts
Our cloning strategies constructs rely mostly on parts available from the registry. New parts have been introduced in the context of the use of the promising future model organism
Vibrio natriegens. This page gives an overview of the newly designed parts we contributed to the iGEM registry, native p1 promoter, improved pTET promoter and parts enabling CRISPR mediated knockout.
BBa_K3171171 Native V. natriegens P1 promoter, constitutive
This part is the native P1 promoter from the
V. natriegens genome. Genes under the control of this promoter are constitutively transcribed by the organism’s ribosomal protein P1. This part was obtained by the sequencing offer of gBLOCKS from IDT. We show that there is a 225 times increase of fluorescence using the P1 promoter in
E. coli compared to
E. coli without a plasmid. At the same time, measurement in
V. natriegens showed a 3 times increase of fluorescence.
Figure 1: Fluorescence of the reporter mCherry under control of the constitutive P1 promoter in V. natriegens and E. coli in comparison to control without plasmid.
BBa_K3171173 Improved Part: Synthetic promoter library on BBa_R0040
Employing a synthetic promoter library (SPL) we optimized the function of tetracycline inducible promoter. The SPL was constructed using a PCR with randomized primers to yield arbitrary sequences in the promoter region. A total of 245 clones were screened to find a target with enhanced functionality. The screening of the final 6 interesting variations revealed clone 6 as the sequence with increased inducibility (Figure 2). We calculated a fold change of 34 comparing fluorescence in the induced and uninduced state! Additionally, this promoter also exhibited a low basal level of fluorescence that is comparable to background fluorescence of
E. coli without any reporter (Figure 3).
Figure 2: Clone 6 exhibits the highest fluorescence fold change upon induction. Single clones from the synthetic promoter library were induced with 250 ng/ml anhydrotetracycline. The fluorescence fold change is calculated by dividing the fluorescence in the induced state by fluorescence in the absence of inducer. The used fluorescence reporter is mCherry.
Figure 3: The basal level of reporter expression is comparable to E. coli without the construct. Clone 6 from the synthetic promoter library was induced with 250 ng/ml anhydrotetracycline. The used fluorescence reporter is mCherry.
CRISPR Parts
The variable components needed for a CRISPR mediated knockout are gRNA and a sequence for homologous recombination. gRNA and the homologous part have to be engineered strictly depending on the target. In this case, all parts were designed with the aim to knockout HisD in
V. natriegens.
The gRNA enables the Cas9 enzyme to recognize and cut the correct cut site. The Cas9 enzyme forms a complex with the gRNA and can then find the target sequence based on complementary base pairing. Parts
BBa_K3171180,
BBa_K3171181 and
BBa_K3171182 have been designed with the help of synthego software that helps to select suitable target site, considering e.g. possible off-targets or location within coding region. These parts have been ordered as single stranded oligonucleotides, annealed and ligated into a vector encoding the necessary compounds of CRISPR machinery.
This part serves as a template for homologous recombination once the Cas9 enzyme has cut the target site. Consequently, it is designed according to the up- and downstream regions of the targeted sequence.
BBa_K3171177 has a total length of 3 kbp, 1.5 kbp for each up- and downstream of HisD, and was ordered as gBLOCKS from IDT’s synthesis offer. 1.5 kbp (
BBa_K3171178) and 600 bp (
BBa_K3171179) length were achieved using PCR and respective primers.
