Difference between revisions of "Part:BBa K4195191"

Latest revision as of 07:41, 12 October 2022

pRha-B0034-EYFP-B0015

This composite part is used to contrast with the improved L-rhamnose-inducible promoter circuit BBa_K4195109.

Biology

pRha

L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions. The genes rhaBAD are organized in one operon which is controlled by the rhaP_BAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaP_RS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaP_BAD and the rhaP_T promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaP_BAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.

Usage

pRha (BBa_K914003) is a strictly regulated promoter, which was firstly registered in 2013 and has been widely used in stringently expression of GOI (gene of interest). And the eyfp gene (BBa_E0030) was chosen as the reporter to finally construct the characterization circuit. Different sub parts of the circuit were assembled into pSB4K5 plasmid backbone using Gibson assembly to get the composite part BBa_K4195191 (Fig. 1). The Gibson assembly reaction mixture was transformed into E. coli DH5α and E. coli BL21(DE3), then the positive colonies were confirmed by colony PCR and sequencing.

Fig. 1 Gene circuit of BBa_K4195191.

Characterization

When we constructed this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1288bp (lane K4195191).

Fig. 2 The result of colony PCR. Plasmid pSB4K5.

Then, the colony with the corrected sequence was cultivated and induced to measure the expression intensity of EYFP compared with BBa_K4195109. Both the composite parts were induced under L-rhamnose whose final concentration was 0.1 mg/mL and the same volume sterile water was added as the control. The fluorescence intensity and OD₆₀₀ was measured in 2 h after induction. The gene circuit with rhaS can produce higher fluorescent signal obviously although there exists little expression leakage. (Fig. 3)

Fig. 3 The comparison of normalized fluorescence intensity between BBa_K4195109 and BBa_K4195191.

Sequence and Features