IPTG inducible promoter with RBS

R0010.B0034

Usage and Biology

R0010 and B0034 will be digested and ligated together in the manner described on the "Registry of Standard Biological Parts" website.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Contribution made by SHSID 2020

Recombinant FnCpf1 production, purification, and SDS-PAGE analysis

The backbone for BBa_K3521005 was derived from pET-28a vector. The recombinant plasmid was transformed into BL21 (DE3) competent cells and induced with IPTG. After induction, a specific protein band that is consistent with the theoretical molecular weight of FnCpf1 was detected in SDS-PAGE (Figure 2). The recombinant FnCpf1 was successfully purified by Ni-affinity chromatography.

Figure 1. pET28a-Cas12a(FnCpf1)

Figure 2. SDS-PAGE analysis of FnCpf1 production in BL21 (DE3) cells. After induction, cells were collected by centrifugation and broken by sonication. The resulting supernatants were loaded to the PAGE gel with different amounts. Lane2 1-4: the samples from the cultures induced with 1 mM IPTG; Lane2 5-8: the samples from the cultures induced with 0.5 IPTG; lane 9: protein molecular weight standards.

Contribution made by Aix-Marseille 2021

IPTG doesn't induce gene transcription

For the goods of our project, we designed a plasmid by cloning BBa_K3788013 in BBa_J04500 ; and transformed it into DH5a E.Coli competent cells.

As we can see green colonies on LB+Agar plates without IPTG induction, we wanted to test if IPTG induction would enhance fluorescence level.

To test the ability of the promoter to be induced by IPTG, we made bacterial liquid cultures containing BBa_J04500+BBa_K3788013 and bacteria containing BBa_J04500. We then incubated our cultures with different concentrations of IPTG for 3 hours.

Table 1. IPTG quantities added to our cultures.

After induction, we measured fluorescence intensity for each condition diluted at different 600OD :

Figure 1. fluorescence intensity obtained for each induction condition. sf : bacteria containing BBa_J04500+BBa_K3788013 ; V : bacteria containing BBa_J04500

From this experiment we can conclude that adding IPTG to the growing medium do not affectate the fluorescence intensity, so we concluded that it doesn’t enhance the protein production.

An hypothesis could be that the cellular LacI quantity is not sufficient to repress the gene transcription ; or that the promoter is not responding to repression and so can’t be induced by IPTG.

Glucose can repress gene transcription

As we didn’t observe any difference due to IPTG induction we decided to test the glucose repression.

To test the ability of the promoter to be repressed by glucose, we made bacterial liquid cultures containing BBa_J04500+BBa_K3788013.

We then incubated our cultures with different concentrations of glucose and measured fluorescence intensity every 60 minutes.

Table 2 :. glucose concentration added to our cultures. explication

Figure 2 :. fluorescence intensity obtained at 0 ; 60 ; 120 and 170 minutes for each condition.

As we can see, the samples with the highest glucose concentration had a lower fluorescence intensity at all times.

Therefore we can conclude that glucose repression of BBa_J04500 is functional.

From the experiments we made, we propose that BBa_J04500 can’t be used as an IPTG inducible promoter, but can be glucose-repressed to control the protein production.