Difference between revisions of "Part:BBa K3037000"

Revision as of 14:48, 21 October 2019

pOCC97 plasmid backbone for expression (optimized)

pOCC97
Function	Expression
Use in	Escherichia coli pRARE T7
RFC standard	RFC 10
Submitted by	Team: TU_Dresden 2019

Overview

The TU Dresden 2019 team designed this BioBrick in order to express its fusion protein (more information).

Features:

IPTG inducible
LacO promoter and LacI inhibitor
Kanamycin resistance
Needs T7 RNA polymerase (a viral RNA polymerase for high expression)
- For optimal results use BBa_K3037000 in combination with a pRARE plasmid, carried by the used Escherichia coli (E. coli) strain
- In case you use pRARE (Cm), you need to grow in a media containing both antibiotics - Kanamycin (Kan) and Chloramphenicol (Cm)
- Alternatively use a strain which can express T7 from their genome, regular E. coli strains do not express T7!
Contains SP6 site which is a commonly used primer site for sequencing the inserted part
Codon optimized for E. coli

Very well established expression plasmid for recombinant proteins in E. coli, 
getting any BioBrick you need from ligation to expression in just 24 hours!

Biology

The lactose operon (lac operon) is a polycistronic bacterial operon that encodes the genes of lactose metabolism. It is consists of three structural genes: a promoter, an operator and a terminator. A bacterial cell synthesizes enzymes involved in lactose metabolism only under two conditions: in the presence of lactose and when the cells is lacking glucose. [1]

The regulation of the lac operon occurs according to the principle of negative feedback: the more lactose is present in the environment - the more enzymes for its catabolism is synthesized (positive direct connection); the more enzymes are present in a cell - the less lactose remains, and finally, the less lactose in the environment - the less enzymes are produced (double negative feedback).[2]

In the absence of lactose in the cell, or at a low concentration, the repressor protein reversibly binds to the operator region and inhibits transcription. The reporter protein is a product of the LacI monocistronic operon. In the absence of lactose in the cell, enzymes for lactose metabolism are not synthesized. Besides, if the glucose concentration in the cell is sufficient to maintain metabolism, activation of the lactose operon also does not occur. The promoter sequence of the lactose operon is weak, therefore, even in the absence of a repressor protein in the operator site, transcription is practically not initiated.

When the concentration of glucose in the cell decreases, the enzyme adenylate cyclase is activated. Glucose is an inhibitor of this enzyme and activates phosphodiesterase, which catalyzes the conversion of the cAMP molecule to AMP. Adenylate cyclase catalyzes the conversion of ATP to the cyclic form - cAMP. cAMP binds to a catabolism activating protein (CAP), and a complex is formed that interacts with the promoter of the lactose operon. It changes its conformation, and increases the affinity of RNA polymerase for this site. In the presence of lactose, expression of the operon genes occurs.[2]

Klaus I. Matthaei, in Handbook of Stem Cells, 2004

Characterization

Outline

Expression of different proteins (BBa_3037003)(BBa_K3037007) with growth curves
Expression of proteins with the plasmid before and after optimization at different temperatures
SDS-PAGEs for the expression assay over the time of Full Construct (BBa_K3037003)
Image analysis of the expression of SDS-PAGEs with ImageJ
Expression of different BioBricks in pOCC97

Experiments in Detail

1) Expression of different proteins (BBa_3037003)(BBa_K3037007) with growth curve

Two different proteins were expressed using this backbone: HRP (BBa_K3037007) and a bigger fusion protein (BBa_K3037003). The constructs were expressed in E. coli pRARE T7.

The results prove that when expressed, none of our two proteins inhibits the growth of the bacteria, yet the growing ratio is lower in the culture that is expressing a bigger protein.

Growth curve (Comparison of growing of s¡different size proteins)
Growth curve (When MBP-HRP (BBa_K3037008) composite BioBrick is expressed)

2) Expression of proteins with the plasmid before and after optimization at different temperatures

To use this backbone as convenient expression vector in iGEM, we had to include the Prefix and Suffix of the BioBrick Assembly. Subsequentially, we compared the expression of the original vector we recieved, pOCC97, to our optimized version for iGEM, BBa_K3730000.

We found that the original plasmid had a XbaI site, which we used to insert our BioBrick BBa_K3037003. This ilegal restriction site was later removed with an overhang PCR. The original XbaI restriction site was positioned downstream of the T7 polymerase promoter and upstream the RBS sequence of the plasmid. This way only BioBricks that already has a RBS fused to them could be expressed. Since we were using the RFC25 standard of Freiburg for our fusion proteins, the inserted protein contained already its own RBS in the Prefix.

However, we experienced on our own how difficult it is to add such a small sequence, as an RBS, to our other constructs. Therefore we redesigned the plasmid to be ready for expression in a single digestion+ligation reaction. We removed the XbaI restriction site and included a Prefix and Suffix of the RFC 10 standard after the RBS of the plasmid.

We used the BioBrick assembly method to insert our BioBrick BBa_K3037003, which has its own RBS due to the RFC 25 standard.

The comparison of the growth curves shows that, although, the new plasmid adapted to the RFC 10 standard didn't affect the growing rate of the bacteria, as the original one, but the expression of the protein was improved.

Comparison of the growth curves of optimized and not optimized pOCC97.

Comparison of the growth curve compared before and after optimization

3) SDS-PAGEs for the expression assay over the time of Full Construct (BBa_K3037003)

Comparison of the expression of MBP-HRP (BBa_K3037008) and Full Construct (BBa_3037003)

SDS-PAGE (Comparison of expression of different proteins in the vector before and after optimization)

Expression of full construct in pOCC97 not optimized at 18ºC

SDS-PAGE (Expression of fusion protein BBa_K3037003 at 37ºC before optimization)

Expression of Full Construct in pOCC97 at 37ºC

SDS-PAGE (Expression of fusion protein BBa_K3037003 at 37ºC before optimization)
SDS-PAGE (Expression of fusion protein BBa_K3037003 at 37ºC before optimization)

Expression of Full Construct in pOCC97 optimized

SDS-PAGE (Expression of fusion protein BBa_K3037003 after optimization)

4) Image analysis of the expression in the SDS-PAGEs with imageJ

Different induction concentration not optimized at 18ºC and at 37ºC

Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

Not optimized at different temperature at 0.2 mM, 0.5 mM and 1 mM IPTG expression induction

Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

Different temperature optimized 1mM

Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

Different induction concentration optimizedat 18 degrees

Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

Comparison optimized vs not optimized

Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

Conclusion

Based on this analysis, it can be concluded that optimal conditions for the expression of our fusion protein, BBa_3037003, are overnight expression at 18ºC while inducing with 0.5 mM IPTG. We are proud to say that our optimized pOCC97 shows a increased expression robustness under varying conditions over time.

5) Expression of different BioBricks in pOCC97

With this experiment we proved that is easy to express different BioBricks functionally with this vector.

Expression of different BioBricks in pOCC97

Sequence

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 5283
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 5289
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 5283
Illegal BglII site found at 5169
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found at 5283
Illegal suffix found at 2
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found at 5283
Plasmid lacks a suffix.
Illegal XbaI site found at 5298
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 342
Illegal NgoMIV site found at 3389
Illegal NgoMIV site found at 3549
Illegal NgoMIV site found at 5137
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal SapI.rc site found at 2468

Design Notes

This BioBrick was designed to fit the RFC 10 standard using the primers:

Forward: tactagtagcggccgctgcagCCGTTATAGAAGCTTGAGTATT

Reverse: gaattcgcggccgcttctagagGCCCATGGATATATCTCCTTCT

References

1. Jacob F; Monod J. Genetic regulatory mechanisms in the synthesis of proteins, J Mol Biol. journal, 1961, vol. 3: p. 318—356.

2. J. Parker, Encyclopedia of Genetics, 2001

3. Klaus I. Matthaei, in Handbook of Stem Cells, 2004 (For picture)

@@ Line 82: / Line 82: @@
 === Experiments in Detail ===
-==== 1) Expression of different proteins, growing curve [https://parts.igem.org/Part:BBa_K3037003 (BBa_3037003)][https://parts.igem.org/Part:BBa_K3037007 (BBa_K3037007)] ====
+==== 1) Expression of different proteins [https://parts.igem.org/Part:BBa_K3037003 (BBa_3037003)][https://parts.igem.org/Part:BBa_K3037007 (BBa_K3037007)] with growth curve ====
 Two different proteins were expressed using this backbone: HRP [https://parts.igem.org/Part:BBa_K3037007 (BBa_K3037007)] and a bigger fusion protein [https://parts.igem.org/Part:BBa_K3037003 (BBa_K3037003).]
+The constructs were expressed in <span style="font-style: italic;">E. coli</span> pRARE T7.
-The organism used for the expression was <span style="font-style: italic;">E. coli</span> pRARE T7
+The results prove that when expressed, none of our two proteins inhibits the growth of the bacteria, yet the growing ratio is lower in the culture that is expressing a bigger protein.
-The results prove that when expressed, non of the two proteins affect the growing curve of the bacteria. Also, it can be seen how the growing ratio is lower in the culture that is expressing a bigger protein.
 <gallery mode="packed", caption="Assay to determine protein amount", widths=400px, heights=200px>
-File:T--TU_Dresden--Expression_of_proteins_BBa_K3037000.png|<span style="color:#0000ff"> ''Growing curve''  </span> (Comparison of growing of s¡different size proteins)
+File:T--TU_Dresden--Expression_of_proteins_BBa_K3037000.png|<span style="color:#0000ff"> ''Growth curve''  </span> (Comparison of growing of s¡different size proteins)
-File:T--TU_Dresden--Expression_MBP_HRP_in_pOCC97.png|<span style="color:#0000ff">''Growing curve'' </span>  (When MBP-HRP [https://parts.igem.org/Part:BBa_K3037008 (BBa_K3037008)] composite BioBrick is expressed)
+File:T--TU_Dresden--Expression_MBP_HRP_in_pOCC97.png|<span style="color:#0000ff">''Growth curve'' </span>  (When MBP-HRP [https://parts.igem.org/Part:BBa_K3037008 (BBa_K3037008)] composite BioBrick is expressed)
 </gallery>
 ==== 2) Expression of proteins with the plasmid before and after optimization at different temperatures ====
-To use this backbone as an expression vector adapted to the iGEM standards, we had to mutate it first to include the Prefox and Suffix of the BioBrick Assembly. Before to do that, we thought that it will be great to have a comparison between the expression before and after the mutation, a process that we called optimization.
+To use this backbone as convenient expression vector in iGEM, we had to include the Prefix and Suffix of the BioBrick Assembly.
+Subsequentially, we compared the expression of the original vector we recieved, pOCC97, to our optimized version for iGEM, BBa_K3730000.
-We found that the original plasmid had a XbaI sequence that we used to insert our BioBrick expressed protein [https://parts.igem.org/Part:BBa_K3037003 BBa_K3037003.] This ilegal restriction site was removed during the optimization process. The XbaI restriction site was placed after the promoter (T7) and before the RBS sequence of the plasmid. However, since we were using the RFC 25 standard for fusion proteins, the inserted protein contained this was its own RBS (included in the prefix of the RFC 25 standard).
+We found that the original plasmid had a XbaI site, which we used to insert our BioBrick [https://parts.igem.org/Part:BBa_K3037003 BBa_K3037003.] This ilegal restriction site was later removed with an overhang PCR. The original XbaI restriction site was positioned downstream of the T7 polymerase promoter and upstream the RBS sequence of the plasmid. This way only BioBricks that already has a RBS fused to them could be expressed. Since we were using the RFC25 standard of Freiburg for our fusion proteins, the inserted protein contained already its own RBS in the Prefix.
-Then, for the optimization we removed the XbaI restriction site and included a Prefix and Suffix of the RFC 10 standard after the RBS of the plasmid. After that we used the BioBrick assembly method to insert there our protein BBa_K3037003, which has its own RBS due to the RFC 25 standard.
+However, we experienced on our own how difficult it is to add such a small sequence, as an RBS, to our other constructs. Therefore we redesigned the plasmid to be ready for expression in a single digestion+ligation reaction.
+We removed the XbaI restriction site and included a Prefix and Suffix of the RFC 10 standard after the RBS of the plasmid.
-The comparison of the growing curve shows that, although, the new plasmid adapted to the RFC 10 standard didn't affect the growing rate of the bacteria, as the original one, but the expression of the protein was improved.
+We used the BioBrick assembly method to insert our BioBrick [https://parts.igem.org/Part:BBa_K3037003 BBa_K3037003,] which has its own RBS due to the RFC 25 standard.
+The comparison of the growth curves shows that, although, the new plasmid adapted to the RFC 10 standard didn't affect the growing rate of the bacteria, as the original one, but the expression of the protein was improved.
 [[File:Growingcurves.png|center|700px|thumb|none|Comparison of the growth curves of optimized and not optimized pOCC97.]]
@@ Line 175: / Line 178: @@
 <b>Conclusion</b>
-Based on this analysis can be concluded that optimal conditions for the expression of [https://parts.igem.org/Part:BBa_K3037003 BBa_3037003] are 18ºC  and 0.5 mM IPTG.
+Based on this analysis, it can be concluded that optimal conditions for the expression of our fusion protein, [https://parts.igem.org/Part:BBa_K3037003 BBa_3037003,] are overnight expression at 18ºC while inducing with 0.5 mM IPTG.
-Also the optimization process is better than the not optimazed as the expression is more stable over time
+We are proud to say that our optimized pOCC97 shows a increased expression robustness under varying conditions over time.
 ==== 5) Expression of different BioBricks in pOCC97 ====
-With this experiment we proved that is easy to express diferent and functional BioBricks with this vector
+With this experiment we proved that is easy to express different BioBricks functionally with this vector.
 [[File:T--TU_Dresden--Expressing_GFP_BBa_K3037000.png|center|400px|thumb|none|Expression of different BioBricks in pOCC97]]