Part:BBa_K2842669

mScarlet reporter with TerL-C intein on the N terminus

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.

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). 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.

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>

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

TESTING

WE improved the part BBa_K1362101 ( PUT LINK HERE !!!!!!!1111) 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 fluoresnence of our reporter protein when driven purely by the leaky 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 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

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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