Parts

Gene cassettes:-

We have made three principal gene cassettes - lactate response cassette (BBa_K3848011), fusion protein secretion cassette (BBa_K3848010) and kill switch cassette (BBa_K3848000) and the AND gate promoter (BBa_K3848002) that responds to hypoxia as well as lactate..

As a proof of concept, we show the experimentation in E. coli chassis but the project relies on the use of Bifidobacterium longum as the chassis organism. And to bolster the credibility of the proof of concept in E. coli, the pMB1 plasmid was chosen for cloning and expression of all our cassettes as pMB1 can also be cloned and expressed in B. longum^[1]. Hence, only the codons need to be optimized for expressing in B. longum.

Lactate response cassette:-

This cassette consists of the genes lldP (permease) (BBa_K3848008) and lldR (repressor) (BBa_K3848009). The lactate response cassette is responsible for uptaking lactate in the environment and performing the required regulatory functions on other cassettes via its lldR gene product and the lldR responding promoters in the respective cassettes. The permease helps rapid uptake of lactate from the environment and the repressor is involved in regulation in other cassettes (mentioned in respective cassette’s section).
We added a constitutive promoter (BBa_J23118) in the cassette, and a RBS (BBa_B0015) at the end to terminate transcription. Hence we have made this part functional for our purposes. The composite part for this cassette is BBa_K3848011.

Fusion protein secretion cassette:-

This cassette is regulated by an AND gate (BBa_K3848002) which responds to hypoxia and lactate concentration. This part of the fusion construct consists of a hypoxia-inducible promoter (BBa_K387003) intercalated between lactate operators (BBa_K1847008). The lldR negatively regulates the AND gate and the depression occurs in the presence of lactate (uptaken by permease).

This composite part also has a new part contributions from our team, and that is the TRAIL-Smac fusion construct (BBa_K3848000) which is the coding region downstream of the AND gate (BBa_K3848002) in this cassette .The cassette containing all these parts has been made into a composite part - TRAIL-Smac fusion peptide secretion cassette (BBa_K3848010).

Kill switch cassette:-

The kill switch cassette designed by our team is related to Team iGEM20_Cornell’s part (BBa_K3419005), but we have made modifications to suit the kill switch to our project. Our kill switch adds a new layer of regulation in the form of the ssRA-LVA tagged cI repressor (BBa_K327018) and cI regulated promoter (BBa_R0051) for finer control of the kill switch. The kill switch that we have made is a composite part in the registry (BBa_K3848004).

List of parts used:-

#	Name	Type	Description	Length (in bp)
1	BBa_J23118	Promoter (constitutive)	A medium strength constitutive promoter.	35
2	BBa_B0034	RBS	Strong RBS for efficient translation.	12
3	BBa_K3848008	Coding (lldP)	Transports L-lactate across the membrane. Can also transport D-lactate and glycolate.	1652
4	BBa_K3848009	Coding (lldR)	A transcription factor that binds lldR operator sites in lactate regulated promoters.	782
5	BBa_B0015	Terminator	Terminator sequence efficient for halting transcription in the forward direction.	129
6	BBa_K1847008	Lactate regulated promoter	Lactate regulated promoter; has operator sites that can be bound by lldR gene product in the absence of lactate, repressing the promoter.	143
7	BBa_B0032	RBS	Medium strength RBS	13
8	BBa_K112807	Coding (T4 antiholin)	T4 antiholin binds holin and prevents holin multimers from forming pores on the inner membrane of bacteria.	311
9	BBa_K327018	Coding (cI repressor)	Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator	750
10	BBa_R0051	cI regulated promoter	The cI regulated promoter is based on the pR promoter from bacteriophage lambda. It is a strong promoter and has two DNA binding sites for the lambda cI repressor mentioned above. cI binding results in repression of transcription.	49
11	BBa_K112805	Coding (holin)	Holins from T4 bacteriophage assemble together to form pores on the inner membrane of bacteria.	12676
12	BBa_K112806	Coding (T4 endolysin)	The lysozyme from enterobacteria phage T4 degrades peptidoglycan layer.	514
13	BBa_K3848002	AND gate promoter	This promoter responds to hypoxia and lactate, it is active under hypoxia and high lactate conditions. Repression occurs by lldR binding to the operators under low lactate conditions.	217
14	BBa_K3848000	Coding (TRAIL-Smac Fusion Protein with HlyA signal peptide)	This fusion protein which can induce apoptosis and kill cancer cells. It has N terminal 7 amino acid residues of Smac/DIABLO (small-mitochondria associated activator of Caspase) and the soluble portion of TRAIL (TNF related Apoptosis Inducing Ligand).	891

List of composite parts made:-

#	Name	Type	Description	Length (in bp)
1	BBa_J23118	Composite	A lactate sensitive kill switch that allows the bacteria to survive only in lactate-rich conditions and a certain amount of time after removal of lactate. Once the antiholin is overwhelmed by holin, the cell killing arm of the kill switch takes over.	2837
2	BBa_K3848010	Composite	This composite part has dual regulation of hypoxia and lactate, both are required for expression of the TRAIL-Smac fusion peptide, which will get secreted out of the cell.	1271
3	BBa_K38480011	Composite	This cassette codes for lactate permease and repressor under a constitutive promoter.	2658

Reference

[1] Rossi M, Brigidi P, Gonzalez Vara y Rodriguez A, Matteuzzi D. Characterization of the plasmid pMB1 from Bifidobacterium longum and its use for shuttle vector construction. Res Microbiol. 1996;147(3):133-143. doi:10.1016/0923-2508(96)80213-0

Team:IISER Bhopal/Parts