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