We have performed the characterization of the eforRED (BBa_K592012) protein expression in correlation with the eforeRED transcript level along time after IPTG induction. Our rt-qPCR study find that under the 1mM IPTG-induced T7 activity, eforRED mRNA expression has an exponential increase form 0-10 hrs and gradually decreases later on. Visible color changes at around 4-6hrs allow us to correlate the level transcripts are required for achieving visible signals under T7 promotor activity; Of note, our data provide a promising tool for estimating the conditions (e.g. time, density of cells) required for accumulating the same mRNA transcript level to attain a visible efoRED signal, if the promotor is changed.

Sequence analysis of RpfC homologs in Xcc and Xcm

Please refer to the detail in Homology Study.

Juxtamembrane peptide alignment:ClustalW alignment of Xcc and Xcm RpfC protein, analysing the 22 amino acid-long N-terminal receptor and conserved regions.

Kinase domain alignment:ClustalW alignment of Xcc and Xcm RpfC protein, its domain organisation and homology.

3D model of RpfC:De Novo peptide prediction of N-terminal receptor using PEP-FOLD 3.5 (RPBS Mobyle platform)

New biobrick

BBa_K3071007: Phage shock protein F transcriptional activation domain (PspF TAD)

BBa_K3071009: Phage shock protein F transcriptional activation domain fused with cAMP receptor-like protein (pspF TAD - Clp)

BBa_K3071010: RNA polymerase Sigma-54 factor (rpoN)

BBa_K3071011: Clp-binding site I from gumB operon (CBS I)

BBa_K3071012: Clp-binding site II from gumB operon (CBS II)

BBa_K3071013: PspA promotor sequence

BBa_K3071014: gumB CBS I & II-regulated pspA promotor

Improvement in documented sequence

All our foreign protein encoding sequence is being codon optimized to suit for the Escherichia Coli expression including RpfC (BBa_K3071000), RpfG (BBa_K3071002) and Clp (BBa_K3071004).

BBa_K1315002: It found to have multiple stop codon at the position 42, 69, 251, 453, 715 in amino acid sequence. We also find that the sequence is actually translated in the lower strand in 5’→3’ direction and recorded as the new biobrick (BBa_K3071000)

BBa_K1315003: Our western blot result indicate this protein encoding sequence is not optimized to be expressed inside E.coli. Our new biobrick (BBa_K3071002) shows a higher expression rate than the old biobrick, which confirm the success of codon optimization.

BBa_K1315005: Sequence optimized and recorded as new biobrick (BBa_K3071004)

Team:Hong Kong-CUHK/Contribution

Contribution

EforRED mRNA expression

Sequence analysis of RpfC homologs in Xcc and Xcm

New biobrick

Improvement in documented sequence