Team:SDU-Denmark/Contribution
Contribution
Abstract
On this page, you can read how we have made a useful contribution to future iGEM teams.
Introduction
Our contribution for future iGEM teams is a new part for the Part Registry (BBa_K4059009) which also includes testing of an existing iGEM part (BBa_I732820). We have created and tested a new inducible promoter system, which is a mutant T7lac promoter. The T7 promoter system allows induction of gene expression with isopropyl-β-D-1-thiogalactopyranoside (IPTG) in E. coli strains with the bacteriophage T7-polymerase in their genome (such as ER2566 or in general BL21(DE3) derivates). The mutant T7lac promoter is to be used together with the lacI, encoding the LacI repressor protein. Therefore, we also tested the function of LacI.
BioBrick design
The promoter sequence is obtained from a scientific article, which has characterised the specific mutant T7lac promoter as the most efficient regarding product yield of Psilocybin. The mutation reduces the strength of the promoter to a medium strength promoter, which is useful since the wild type T7lac promoter tends to overburden cells. This is due to its high strength and thereby a very high expression of extrinsic mRNA at the expense of intrinsic mRNA. The leaky expression of the genes controlled by wild type T7lac promoter is potentially caused by the high strength. A high strength promoter may not necessarily be a problem when aiming to express high amounts of a single protein for purification etc. Especially, when aiming to express enzymes for a synthetisation pathway, it is preferable to have a more moderate gene expression, since there are often multiple enzymes involved. In this case, a medium strength inducible T7lac promoter will be very useful. [1]
Our part with the mutant T7lac promoter contains a Ribosomal Binding Site (RBS), which prepares the brick for direct assembling with desired genes.
How it works
In brief, the mutant T7lac promoter system has two ways of repressing gene transcription. First of all, the transcription of genes controlled by T7lac promoter can only be performed by a specific polymerase, T7-polymerase (originates from bacteriophages). The promoter for the expression of T7-polymerase (in genomic DNA) contains a Lac operator site, to which the repressor protein LacI binds and inhibits T7-transcription. Second, the mutant T7lac promoter itself contains a lac operator site, to which LacI also binds and inhibits the transcription of related genes. When IPTG is added, the LacI dissociates from the Lac operator sites, allowing the expression of T7-polymerase and thereby the transcription of the introduced genes. [2] Please read more at experiments.
Characterisation
In our project, the mutant T7lac promoter is combined with 5 of our genes (PsiD, PsiK, PsiM, PsiH and CPR) and placed into a plasmid containing LacI. The expression of the genes controlled by the mutant T7lac promoter is tested using Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR). The results shows that the gene expression is induced by IPTG (figure 1 + 2). It increases over time and the highest expression is seen 2 hours post-induction. The induction also confirms the functionality of the LacI used (BBa_I732820), since our ER2566 strain does not naturally over express LacI. Therefore, the addition of a functional LacI by cloning is necessary for the T7lac promoter to be sufficiently repressed in the absence of IPTG.
Figure 1: RT-qPCR of PsiD, PsiK and PsiM in ER2566 cells. The graph shows the gene expression of PsiD, PsiK and PsiM measured before inducing with IPTG, 1 hour and 2 hours after induction with IPTG. The gene expression of PsiD, PsiK and PsiM are normalized to the housekeeping gene GyrA. The y-axis shows the relative expression level.
Figure 2: RT-qPCR of PsiH N-terminal in ER2566 cells. The graph shows the gene expression of PsiH measured before inducing with IPTG, 1 hour and 2 hours after induction with IPTG. The gene expression of PsiH is normalized to the housekeeping genes GyrA (yellow) and Rpo (orange). The y-axis shows the relative expression level.
How to use it
Our mutant T7lac promoter part should be inserted into the desired backbone in front of the desired gene, which does not already have an RBS. It must be used in a strain, which expresses the T7-polymerase, controlled by a promoter with a Lac operator. The strain should also have an overexpression of LacI; this can be obtained either by using a special E. coli strain (such as BL21(D3)), which already overexpresses LacI AND contains the T7-polymerase gene; or by cloning another T7-polymerase containing strain (such as ER2566) with an extra LacI gene in a plasmid (together with T7lac promoter system or on a separate plasmid).
Below is an illustration of how to assemble the promoter with the desired gene.
Figure 3: Illustration of the lacI IPTG inducibal promotor system used in BL21 E. coli which co-expresse T7 polymerase.
When having confirmed the cloned plasmid, the bacteria strain is ready to use. The induction is performed when culture has been grown until the mid-exponential phase (OD600 = 0.3-0.5) by adding IPTG to final concentrations between 0.1mM – 1mM. Our results showed optimal gene expression after 2 hours of induction, but this may vary depending on growth conditions, bacteria strain and expressed gene(s).
References
Administration
