Difference between revisions of "Part:BBa K2842669"
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This DNA construct encodes a C terminal segment of the AceL-TerL intein fused to the N terminus of a mScarlet reporter protein with a C terminal StrepTag for purification. The AceL-TerL intein acts as a protein ligase that forms a peptide bond between this protein and another protein bound to a complementary split intein, during this process the intein splices itself out of protein and is not present in the end fusion protein. This construct is a modular platform for the creation of split intein fusion proteins through the SapI restriction sites located immediately upstream and downstream of the mScarlet reporter. BBa_K2832680 was designed to be used in conjunction with this construct as it contains the corresponding N terminal intein segment to enable intein trans-splicing.
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This DNA construct encodes a C terminal segment of the AceL-TerL intein fused to the N terminus of a mScarlet reporter protein which contains a C terminal StrepTag for purification. The AceL-TerL intein acts as a protein ligase that forms a peptide bond between mScarlet and another protein bound to a complementary split intein. During this process the intein splices itself out of protein and is not present in the final fusion protein. This construct is a modular platform for the creation of split intein fusion proteins through the SapI restriction sites located immediately upstream and downstream of the mScarlet reporter.BBa_K2832680 was designed to be used in conjunction with this construct as it contains the corresponding N terminal intein segment to enable intein trans-splicing.
 
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We measured the expression of Intein Passenger with a BMG plate reader by analysis of it's fluorescence at varying IPTG concentrations. We found on this that between 400 uM and 800 uM IPTG induction had little effect on expression or on cell growth rate (Fig1A,Fig1B).
 
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We also tested the modularity of the construct by cloning in two new sequences to create the biobricks "NikR" and "ER" which can have been sequenced to confirm the sucessful assembly. This can also be seen in the analytic digest in Figure 2.
 
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[[File:T--UCL--IP_IPTG_Variations.png|400px|thumb|center|
 
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<center>'''Figure 1: Expression of IP at varying IPTG concentrations'''</center>
 
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Initial experiments suggested that 400 uM of IPTG was optimal for expression(data not shown). A plate reader was used to examine the region of IPTG concentrations around 400 uM
 
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[[File: UCLImprovedPartComparison.png |400px|thumb|left|
 
[[File: UCLImprovedPartComparison.png |400px|thumb|left|
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<br><h3>Experimental Results</h3>
 
<br><h3>Experimental Results</h3>
  
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<p> We improved the part [https://parts.igem.org/Part:BBa_K1362101 BBa_K1362101] with this construct. Both parts were designed to allow for the modular design and assembly of intein fusion proteins, but ours has distinct advantages and improvements. We used mScarlet as our reporter instead of mRFP1 as it is known to be brighter than mRFP1 and a fast folder. The result of changing this protein can be seen in the increased fluorescence of our reporter protein when driven purely by the leaky expression from the T7 promoter(Fig3).
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<p> This construct (BBa_K2842669) is also an improvement of the part [https://parts.igem.org/Part:BBa_K2842669 BBa_K1362101]. Both parts were designed to allow for the modular design and assembly of intein fusion proteins, but our part has distinct advantages and improvements. We used mScarlet as our reporter instead of mRFP1 as it is known to be brighter than mRFP1 and a fast folder. Our reporter protein mScarlet has an 11-fold  increase in fluorescence/OD600 compared to BBa_K1362101 when driven by the expression from the T7 promoter (Fig3). Our construct also has a dual function as both an assembly construct and reporter for building new inteins but as an intein fusion by itself. Due to this, it can be used for both to test intein functionality in other constructs and to build new ones.
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Our construct also has a dual function as both an assembly construct and reporter for building new inteins but as an intein fusion by itself. due to this it can be used both to test intein functionality in other constructs and to build new ones.
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*edited to show relevant bands
 
*edited to show relevant bands
 
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We measured the expression of BBa_K2842669 with a BMG Fluostar plate reader by analysis of it's fluorescence at varying IPTG concentrations. We found that mScarlet is very well expressed  when produced from an IPTG-inducible T7 promoter.  Increasing IPTG concentrations from  400 uM to 800 uM  had little effect on expression levels and cell growth rate (Fig1A, Fig1B), indicating that IPTG concentrations at 400 uM are sufficient to overproduce our protein. We also tested the modularity of the construct by replacing mScarlet with with two new sequences to create the biobricks [https://parts.igem.org/Part:BBa_K2842710 BBa_K2842710] and [https://parts.igem.org/Part:BBa_K2842720 BBa_K2842720]. We have confirmed this assembly with Sanger sequencing.
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[[File:T--UCL--IP_IPTG_Variations.png|400px|thumb|left|
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<center>'''Figure 1: Expression of IP at varying IPTG concentrations'''</center>
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Initial experiments suggested that 400 uM of IPTG was optimal for expression(data not shown). A plate reader was used to examine the region of IPTG concentrations around 400 uM
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
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Revision as of 17:22, 17 October 2018


mScarlet reporter with TerL-C intein on the N terminus

mScarlet with TerL-C intein
Function Standardised blue-white screening
Use in E. coli cells
Chassis Tested DH5α cells
Abstraction Hierarchy Composite Device
Related Device BBa_K1362101
RFC standard RFC10,RFC21,RFC23
& RFC25 compatible
Backbone pSB1C3
Submitted by [http://2018.igem.org/Team:UCL UCL iGEM 2018]

This DNA construct encodes a C terminal segment of the AceL-TerL intein fused to the N terminus of a mScarlet reporter protein which contains a C terminal StrepTag for purification. The AceL-TerL intein acts as a protein ligase that forms a peptide bond between mScarlet and another protein bound to a complementary split intein. During this process the intein splices itself out of protein and is not present in the final fusion protein. This construct is a modular platform for the creation of split intein fusion proteins through the SapI restriction sites located immediately upstream and downstream of the mScarlet reporter.BBa_K2832680 was designed to be used in conjunction with this construct as it contains the corresponding N terminal intein segment to enable intein trans-splicing.

Comparisons of fluorescence/OD600 of BBa_K2842669 and BBa_K1362101

(1-Red) BBa_K2842669 in BL21* cells induced with 400 μM IPTG
(2-Orange) BBa_K2842669 in BL21* cells without IPTG induction
(3-Grey) BBa_K1362101 expressed in BL21* cells />


Experimental Results

This construct (BBa_K2842669) is also an improvement of the part BBa_K1362101. Both parts were designed to allow for the modular design and assembly of intein fusion proteins, but our part has distinct advantages and improvements. We used mScarlet as our reporter instead of mRFP1 as it is known to be brighter than mRFP1 and a fast folder. Our reporter protein mScarlet has an 11-fold increase in fluorescence/OD600 compared to BBa_K1362101 when driven by the expression from the T7 promoter (Fig3). Our construct also has a dual function as both an assembly construct and reporter for building new inteins but as an intein fusion by itself. Due to this, it can be used for both to test intein functionality in other constructs and to build new ones.


Figure 1: BsaI digestions

(1) BsaI digested Intein Passenger BBa_K2842669
(2) BsaI digested RFP inteins BBa_K2842680
(3) BsaI digested GFP inteins BBa_K2842690
*edited to show relevant bands

We measured the expression of BBa_K2842669 with a BMG Fluostar plate reader by analysis of it's fluorescence at varying IPTG concentrations. We found that mScarlet is very well expressed when produced from an IPTG-inducible T7 promoter. Increasing IPTG concentrations from 400 uM to 800 uM had little effect on expression levels and cell growth rate (Fig1A, Fig1B), indicating that IPTG concentrations at 400 uM are sufficient to overproduce our protein. We also tested the modularity of the construct by replacing mScarlet with with two new sequences to create the biobricks BBa_K2842710 and BBa_K2842720. We have confirmed this assembly with Sanger sequencing.

Figure 1: Expression of IP at varying IPTG concentrations
Initial experiments suggested that 400 uM of IPTG was optimal for expression(data not shown). A plate reader was used to examine the region of IPTG concentrations around 400 uM </p>


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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 941
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1224
    Illegal BsaI.rc site found at 28
    Illegal SapI site found at 1130
    Illegal SapI.rc site found at 419