Difference between revisions of "Part:BBa K4059009:Design"

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===Design Notes===
 
===Design Notes===
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This is a inducible promoter system, which is a mutant T7 Lac 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 T7 Lac promoter is to be used together with the lacI, encoding the LacI repressor protein.
+
BioBrick design:
−
Consideration: There is choosen a medium inducible promoter system based on the former litterature where a high inducible promoter will not work in the cells.
+
The promoter sequence is obtained from a scientific article, which has characterized 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 preferrable 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. (https://www.nature.com/articles/srep11301).  
 +
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-transscription. 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 (https://pubmed.ncbi.nlm.nih.gov/1902522/). 
  
 
Characterisation:  
 
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. 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 overexpress 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.
+
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 overexpress 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.  
 +
 
 +
[insert 'gene expression' grøn]
 +
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. 
 +
 
 +
[insert 'gene expression' orange]
 +
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).
 +
 
 +
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).
 +
 
 
To read more about the results and the figures; go to https://2021.igem.org/Team:SDU-Denmark/Results
 
To read more about the results and the figures; go to https://2021.igem.org/Team:SDU-Denmark/Results
  

Revision as of 03:06, 22 October 2021


T7Lac Promotor and RBS


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]


Design Notes

BioBrick design: The promoter sequence is obtained from a scientific article, which has characterized 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 preferrable 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. (https://www.nature.com/articles/srep11301). 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-transscription. 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 (https://pubmed.ncbi.nlm.nih.gov/1902522/).

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 overexpress 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.

[insert 'gene expression' grøn] 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.

[insert 'gene expression' orange] 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).

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).

To read more about the results and the figures; go to https://2021.igem.org/Team:SDU-Denmark/Results

Source

Source: https://pubmed.ncbi.nlm.nih.gov/31550507/ and https://www.nature.com/articles/srep11301

References