Overview

This year, our project involved the creation of a wide variety of genetic parts, which served a plethora of purposes. For both the senders' and the receivers' genetic circuits, our team designed new basic parts but also new composite parts. Furthermore, we created a collection of synthetic RBSs, since they were a big part of our project, which we hope can be an essential contribution to future teams. Our collection also includes some pre-existing parts that can already be found in the registry as well as plasmids and primers necessary for our experiments. Finally, we have added the original part we improved, the improved part and all the other parts used in the process of the improvement.

Basic Parts

First, we would like to present the new basic parts that we created. These are RBS sequences that were used to simulate the weights of a perceptron in the senders' population, the main genes that we used for the weighted sum but also regulatory elements.

Name	Type	Description	Designer	Length(bp)
BBa_K4294301	Regulatory	Promoter LuxR-Phlf hybrid	Aristotelis Anastopoulos	55
BBa_K4294150	Regulatory	PlacI Promoter	E.coli genome	54
BBa_K4294998	Regulatory	PlacI Promoter with tet1 RBS	Meyer et. al	82
BBa_K4294190	RBS	tet1 RBS from Marionette Strains	Meyer et. al	28
BBa_K4294004	RBS	Synthetic RBS Ath4: A synthetic ribosome binding site for prokaryotic protein expression, built with the guidance of the RBS calculator and characterized by our team.	Manos Serlis	29
BBa_K4294005	RBS	Synthetic RBS Ath5: A synthetic ribosome binding site for prokaryotic protein expression, built with the guidance of the RBS calculator and characterized by our team.	Manos Serlis	29
BBa_K4294010	RBS	Synthetic RBS Ath10: A synthetic ribosome binding site for prokaryotic protein expression, built with the guidance of the RBS calculator and characterized by our team.	Manos Serlis	29
BBa_K4294011	RBS	Synthetic RBS Ath11: A synthetic ribosome binding site for prokaryotic protein expression, built with the guidance of the RBS calculator and characterized by our team.	Manos Serlis	29
BBa_K4294012	RBS	Synthetic RBS Ath12: A synthetic ribosome binding site for prokaryotic protein expression, built with the guidance of the RBS calculator and characterized by our team.	Manos Serlis	28
BBa_K4294013	RBS	Synthetic RBS Ath13: A synthetic ribosome binding site for prokaryotic protein expression, built with the guidance of the RBS calculator and characterized by our team.	Manos Serlis	29
BBa_K4294102	RBS	BCD2	Mutalik et. al	88
BBa_K4294114	RBS	BCD14	Mutalik et. al	88
BBa_K4294210	Coding	LuxI codon optimised for E Coli (+6His Tag)	Annie Mais, Marianna Mitsioni	615
BBa_K4294211	Coding	LuxI and mNeonGreen coding sequences seperated by the stop-start pentanucleotide "TAATG"	Sofia Oikonomou	1325
BBa_K4294215	Coding	TetR Repressor, Codon Optimized for E.coli	Sofia Oikonomou, Marianna Mitsioni	627
BBa_K4294220	Coding	TetX enzyme	Annie Mais	1167
BBa_K4294401	Coding	LuxR Activator/Repressor Codon Optimised for E Coli	Sofia Oikonomou	756
BBa_K4294410	Coding	mNeonGreen Codon Optimised for E Coli	Sofia Oikonomou	714
BBa_K4294420	Coding	PhlF repressor with a NDENYALAA degradation tag	Aristotelis Anastopoulos	633
BBa_K4294999	Other	Spacer for separating TUs	Aristotelis Anastopoulos	68
BBa_K4294207	Coding	36nt LuxI - sfGFP fusion reporter	Aristotelis Anastopoulos	753

Existing Parts Used

Moreover, we collected and listed the parts that already existed in the iGEM registry and were used by our team.

Name	Type	Short Description	Length(bp)	Group
BBa_J23105	Regulatory	Anderson Promoter (constitutive promoter family member)	35	iGEM2006_Berkeley
BBa_J23106	Regulatory	Anderson Promoter (constitutive promoter family member)	35	iGEM2006_Berkeley
BBa_K3424010	Regulatory	TetR promoter	76	iGEM20_UFlorida
BBa_R0062	Regulatory	Promoter (luxR & HSL regulated -- lux pR)	55	Antiquity
BBa_J107103	Regulatory	LuxR-repressible promoter library member	36	Magni_UNIPV_Lab
BBa_J428046	Regulatory	ptac	56	2021_Engineering_Committee
BBa_J428034	RBS	BCD1	84	2021_Engineering_Committee
BBa_K2680561	RBS	BCD8	84	iGEM18_William_and_Mary
BBa_K2680529	RBS	BCD12	84	iGEM18_William_and_Mary
BBa_B0034	RBS	RBS (Elowitz 1999)	12	Antiquity
BBa_B0015	Terminator	Double Terminator	129	Antiquity

Composite Parts

We continue with the presentation of the composite parts. Here the new parts have been divided according to the population in which they were used, i.e. those concerning the population of senders and the population of receivers.

Senders

Name	Type	Description	Designer	Length(bp)
BBa_K4294704	Composite	LuxI Transcriptional unit with the Ath4 Synthetic RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	831
BBa_K4294705	Composite	LuxI Transcriptional unit with the Ath5 Synthetic RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	831
BBa_K4294710	Composite	LuxI Transcriptional unit with the Ath10 Synthetic RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	831
BBa_K4294711	Composite	LuxI Transcriptional unit with the Ath11 Synthetic RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	831
BBa_K4294712	Composite	LuxI Transcriptional unit with the Ath12 Synthetic RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	830
BBa_K4294713	Composite	LuxI Transcriptional unit with the Ath13 Synthetic RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	831
BBa_K4294721	Composite	LuxI Transcriptional unit with BCD1 RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	886
BBa_K4294722	Composite	LuxI Transcriptional unit with BCD2 RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	890
BBa_K4294723	Composite	LuxI Transcriptional unit with BCD8 RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	886
BBa_K4294724	Composite	LuxI Transcriptional unit with BCD12 RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	886
BBa_K4294725	Composite	LuxI Transcriptional unit with BCD14 RBS (TetR Regulated)	Annie Mais, Panteleimon Marios Stragalis	890
BBa_K4294981	Composite	LuxI_mNeonGreen TU for Ath4 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294982	Composite	LuxI_mNeonGreen TU for Ath5 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294983	Composite	LuxI_mNeonGreen TU for Ath10 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294984	Composite	LuxI_mNeonGreen TU for Ath11 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294985	Composite	LuxI_mNeonGreen TU for Ath12 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294986	Composite	LuxI_mNeonGreen TU for Ath13 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294987	Composite	LuxI_mNeonGreen TU for BCD1 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294988	Composite	LuxI_mNeonGreen TU for BCD2 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294989	Composite	LuxI_mNeonGreen TU for BCD8 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294990	Composite	LuxI_mNeonGreen TU for BCD12 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294991	Composite	LuxI_mNeonGreen TU for BCD14 RBS characterization	Annie Mais, Sofia Oikonomou	1563
BBa_K4294754	Composite	36ntLuxI - sfGFP Transcriptional unit with the Ath4 Synthetic RBS (TetR Regulated)	Aristotelis Anastopoulos	969
BBa_K4294755	Composite	36ntLuxI -sfGFP Transcriptional unit with the Ath5 Synthetic RBS (TetR Regulated)	Aristotelis Anastopoulos	969
BBa_K4294760	Composite	36ntLuxI-sfGFP Transcriptional unit with the Ath10 Synthetic RBS (TetR Regulated)	Aristotelis Anastopoulos	969
BBa_K4294761	Composite	36ntLuxI-sfGFP Transcriptional unit with the Ath11 Synthetic RBS (TetR Regulated)	Aristotelis Anastopoulos	969
BBa_K4294762	Composite	36ntLuxI-sfGFP Transcriptional unit with the Ath12 Synthetic RBS (TetR Regulated)	Aristotelis Anastopoulos	968
BBa_K4294763	Composite	36ntLuxI-sfGFP Transcriptional unit with Ath13 Synthetic RBS (TetR Regulated)	Aristotelis Anastopoulos	969
BBa_K4294771	Composite	36ntLuxI-sfGFP Transcriptional unit with the BCD1 RBS (TetR Regulated)	Aristotelis Anastopoulos	1024
BBa_K4294772	Composite	36ntLuxI-sfGFP Transcriptional unit with BCD2 RBS (TetR Regulated)	Aristotelis Anastopoulos	1028
BBa_K4294773	Composite	36ntLuxI-sfGFP Transcriptional unit with the BCD8 RBS (TetR Regulated)	Aristotelis Anastopoulos	1024
BBa_K4294774	Composite	36ntLuxI-sfGFP Transcriptional unit with BCD12 RBS (TetR Regulated)	Aristotelis Anastopoulos	1024
BBa_K4294775	Composite	36ntLuxI-sfGFP Transcriptional unit with BCD14 RBS (TetR Regulated)	Aristotelis Anastopoulos	1028
BBa_K4294790	Composite	TetX TU	Annie Mais	1364
BBa_K4294780	Composite	TetR TU	Sofia Oikonomou	803

Receivers

Name	Type	Description	Designer	Length(bp)
BBa_K4294801	Composite	OpenLoop	Sofia Oikonomou	1910
BBa_K4294802	Composite	PF	Sofia Oikonomou	1930
BBa_K4294803	Composite	PFc	Sofia Oikonomou	2843
BBa_K4294804	Composite	PFR	Aristotelis Anastopoulos	2808
BBa_K4294805	Composite	PFc+R	Aristotelis Anastopoulos	3864
BBa_K4294806	Composite	TU pLux Output for OL, PF, PFc	Aristotelis Anastopoulos, Sofia Oikonomou	965
BBa_K4294812	Composite	TU Output for PF+ R, PFc+ R	Aristotelis Anastopoulos	932
BBa_K4294807	Composite	TU pLuxr PhlF	Aristotelis Anastopoulos	824
BBa_K4294808	Composite	TU constitutive LuxR for OL	Aristotelis Anastopoulos, Sofia Oikonomou	954
BBa_K4294811	Composite	TU constitutive LuxR for PFc, PFc+R	Aristotelis Anastopoulos, Sofia Oikonomou	954
BBa_K4294809	Composite	TU pLux LuxR PF, PF+ R	Aristotelis Anastopoulos, Sofia Oikonomou	973
BBa_K4294810	Composite	TU constitutive LuxR for PFc, PFc+R	Aristotelis Anastopoulos, Sofia Oikonomou	973

Improvement

Here are also listed the parts considering the “improvement of a part” but you can always find more information about them in the corresponding section of our wiki.

The improved part is:

BioBrick	Type	Short Description	Designer	Length
BBa_K4294559	RBS	Elowitz RBS Version 59 with modified spacer region	Manos Serlis	27

The improvement is upon the part:

BioBrick	Type	Short Description	Designer	Length
BBa_B0034	RBS	RBS (Elowitz 1999)	Antiquity	12

The parts that our wet lab team used, for the improvement experiments, are the:

Name	Type	Description	Designer	Length(bp)
BBa_K4294201	CDS	sfGFP	Aristotelis Anastopoulos, Manos Serlis	717
BBa_K4294203	CDS	36nt PA - sfGFP	Aristotelis Anastopoulos, Manos Serlis	753
BBa_K4294204	CDS	36nt PMK - sfGFP	Aristotelis Anastopoulos, Manos Serlis	753
BBa_K4294205	CDS	36nt AraC - sfGFP	Aristotelis Anastopoulos, Manos Serlis	753
BBa_K4294910	RBS	RBS (Elowitz 1999)	sfGFP TU (Ptet Promoter, B0034 RBS)	942
BBa_K4294911	Composite	36nt PA-sfGFP TU (Ptet Promoter, B0034 RBS)	Aristotelis Anastopoulos, Manos Serlis	978
BBa_K4294912	Composite	36ntPMK-sfGFP TU(Ptet Promoter, B0034 RBS)	Aristotelis Anastopoulos, Manos Serlis	978
BBa_K4294913	Composite	36nt AraC - sfGFP TU(Ptet Promoter, B0034 RBS)	Aristotelis Anastopoulos, Manos Serlis	978
BBa_K4294920	Composite	sfGFP TU (Ptet - v59)	Aristotelis Anastopoulos, Manos Serlis	957
BBa_K4294921	Composite	36nt PA-sfGFP TU(Ptet Promoter, v59 RBS)	Aristotelis Anastopoulos, Manos Serlis	993
BBa_K4294922	Composite	36nt PMK - sfGFP (Ptet Promoter, v59 RBS)	Aristotelis Anastopoulos, Manos Serlis	993
BBa_K4294923	Composite	36nt AraC - sfGFP TU(Ptet Promoter, v59 RBS)	Aristotelis Anastopoulos, Manos Serlis	993
BBa_K4294910	Composite	sfGFP TU (Ptet Promoter, B0034 RBS)	Aristotelis Anastopoulos, Manos Serlis	942

Plasmid Destination Vectors

These are the plasmids we used as vectors to form all of our constructs and are suitable for Golden Gate assembly. For more information about the decision of the plasmid vectors, please refer to the Design tab.

Name	Description	Origin	DBackbone size (bp)
BBa_K4294091	BBa_K4294091	Paul Freemont Lab, EcoFlex MoClo Kit, Addgene	2532
BBa_K4294092	BBa_K4294092	Paul Freemont Lab, EcoFlex MoClo Kit, Addgene	3140
BBa_K4294093	BBa_K4294093	Paul Freemont Lab, EcoFlex MoClo Kit, Addgene	2024
BBa_K4294094	BBa_K4294094	David Savage Lab, Addgene	3712

Primers

In order to build some of our parts, amplify some of the regions of our constructs for verification purposes or even amplify some of our BioBricks to increase their quantity for our experiments we had to design some primers.

Senders and Receivers

Name	Short description	Designer	Length (bp)
BBa_K4294021	Biobrick Prefix Forward	-	20
BBa_K4294022	Biobrick Suffix Reverse	-	21
BBa_K4294031	pTU1 Reverse for Colony PCR	Annie Mais, Sofia Oikonomou	20
BBa_K4294032	pTU2 Reverse for Colony PCR	Annie Mais, Sofia Oikonomou	20
BBa_K4294041	PlacI-tet1 1st primer	Aristotelis Anastopoulos	41
BBa_K4294042	PlacI-tet1 2nd primer	Aristotelis Anastopoulos	34
BBa_K4294043	PlacI-tet1 3rd primer	Aristotelis Anastopoulos	37
BBa_K4294044	PlacI-tet1 4th primer	Aristotelis Anastopoulos	49
BBa_K4294051	TetR-B0015 isolation from plasmid Forward	Sofia Oikonomou	35
BBa_K4294052	TetR-B0015 isolation from plasmid Reverse	Aristotelis Anastopoulos	38
BBa_K4294061	36nt LuxI oligo Forward primer	Aristotelis Anastopoulos	49
BBa_K4294062	36nt LuxI oligo Reverse primer	Aristotelis Anastopoulos	49
BBa_K4294033	pTKEI F for colony PCR	Sofia Oikonomou	20
BBa_K4294034	pTKEI R for colony PCR	Sofia Oikonomou	20

Improvement of A Part

Name	Short description	Designer	Length (bp)
BBa_K4294071	36nt PA oligo Forward primer	Aristotelis Anastopoulos	53
BBa_K4294072	36nt PA oligo Reverse primer	Aristotelis Anastopoulos	49
BBa_K4294073	36nt PMK oligo Forward primer	Aristotelis Anastopoulos	25
BBa_K4294074	36nt PMK oligo Reverse primer	Aristotelis Anastopoulos	54
BBa_K4294075	36nt AraC oligo Forward primer	Aristotelis Anastopoulos	28
BBa_K4294076	36nt AraC oligo Reverse primer	Aristotelis Anastopoulos	54
BBa_K4294077	vv59 RBS oligo Forward primer	Aristotelis Anastopoulos	59
BBa_K4294078	v59 RBS oligo Reverse primer	Aristotelis Anastopoulos	28
BBa_K4294081	Forward primer for sfGFP	Aristotelis Anastopoulos	37
BBa_K4294082	Forward primer for sfGFP fusions	Aristotelis Anastopoulos	36
BBa_K4294083	Reverse primer for sfGFP	Aristotelis Anastopoulos	-

Golden Gate Overhangs

Receivers’ Circuits

For the construction of the Receivers’ Circuits Golden Gate Assembly with BsmBI was performed. The overhangs and the parts are summated in the following table.

Overhang Code And Sequence	TUs	Part Code
A: ATCT	A-TU_pLux_Output_(OL,PF,PFc)-C	BBa_K4294806
B: TTAG	A-TU_pLuxr_PhlF_(PF+R,PFc+R)-E	BBa_K4294807
C: GTCA	C-TU_const_LuxR_(OL)-B	BBa_K4294808
D: GCAA	C-TU_pLux_LuxR_(PF,PF+R)-B	BBa_K4294809
E: CGTA	C-TU_pLux_LuxR_(PFc,PFc+R)-D	BBa_K4294810
	D-TU_const_LuxR_(PFc,PFc+R)-B	54BBa_K4294811
	E-TU_Output_(PF+R,PFc+R)-C	BBa_K4294812

Senders’ Circuits

Senders’ Level 1 TUs were assembled using Golden Gate Assembly with BsaI.

Overhang	Junction
CTAT	pTU1 vector - Promoter
TACT	Promoter - RBS
AATG	RBS - CDS
GCTT	CDS - Terminator
TGTT	Terminator - pTU1 vector
CAAA	36nt LuxI oligo - CDS (sfGFP)
AATG	RBS - 36nt LuxI oligo

Senders’ Level 2 TUs were assembled using Golden Gate Assembly with BsmBI.

Overhang	Junction
ATCT	pTU2 vector - TU from pTU1
TGCC	TU from pTU1 - TetR TU
TTAG	TetR TU - pTU2 vector
TGCC	TU from pTU1 - [PlacI-tet1]
TATG	[PlacI-tet1] - TetR+Terminator from plasmid