Rock-paper-scissors - Scissors primary plasmid
Composite part, consisting of BBa_K180018 and BBa_K180019, but with BBa_K180018 reading along the reverse strand, and BBa_K180019 reading along the forward strand. Forms the 'scissors' primary plasmid in Team:Southampton's rock-paper-scissors project design.
BBa_K180018 reading along reverse strand, and BBa_K180019 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180018) has no effect over mBanana or LuxI expression in BBa_K180019.
See relevant biobrick component parts (BBa_K180018 and BBa_K180019).
Composite part, consisting of BBa_K180005 and BBa_K180006, but with BBa_K180005 reading along reverse strand, and BBa_K180006 reading along the forward strand.
BBa_K180005 reading along reverse strand, and BBa_K180006 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180005) has no effect over LacI or GFP-LVA expression in BBa_K180006.
See relevant biobrick component parts (BBa_K180005 and BBa_K180006)
This part contains a bidirectional, high efficiency terminator, in the BioBytes universal plasmid pAB.See the BioBytes RFC for details on the BioBytes assembly method.
A terminator with long sticky ends can be prepared from this plasmid and assembled downstream of a gene using the Biobytes method. The addition of one DNA segment with the BioBytes method takes only 20 minutes. This ability to easily 'mix and match' terminators and genes allows rapid testing of different terminator designs.
The terminator is derived from part b1006. pAB is entered as part K187000.
Bidirectional transcriptional terminator consisting of a 22 bp stem-loop.
In the naturally-occuring sequence there is a mismatch in the stem of the stem loop. This can be corrected via an A->G mutation (at position 40 -- sequence coordinate/not MFOLD coordinate). The above sequence does not reflect this mutation (but the MFOLD image does). This terminator's location cannot be found using some inverted repeat detectors like PALINDROME because it is too short and contains a mismatch. This one was found with the help of Tom Knight. It lies between two coding regions that point towards eachother.
Derived from luxICDABEG operon terminator of Vibrio fischeri <genbank>AF170104</genbank>.
Rock-paper-scissors - Paper primary plasmid
Composite part, consisting of BBa_K180005 and BBa_K180015, but with BBa_K180005 reading along the reverse strand, and BBa_K180015 reading along the forward strand. Forms the 'paper' primary plasmid in Team:Southampton's rock-paper-scissors project design.
BBa_K180005 reading along reverse strand, and BBa_K180015 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180005) has no effect over GFP or RhlI expression in BBa_K180015.
See relevant biobrick component parts (BBa_K180011 and BBa_K180012).
Two T7 promoters are reversely overlapping.
It is difficult to make this short DNA by PCR as both sides are T7 promoters, which are very similar. It is highly possible that they form loops before they can be synthesized by PCR. Thus we order two single stranded DNA and then anneal them together by touchdown PCR.
The sequence of T7 promoter is TAATACGACTCACTATA.
double terminator (B0012-B0011), reversed
Bidirectional transcriptional terminator cloned in the reverse direction of B0011. 22 bp stem-loop
The RBS of CI434 is CGAGACC in this bistble switch, which makes the RFP expresses three times as the GFP does in this bistable switch.
The library is constructed by mutagenesis.
Binary signal generator, RBS(reverse) - attB - Promoter - attP - RBS
Dual phase protein generator promoter reversed, RBS(reverse) - attB - Promoter(reverse) - attP - RBS This part is composed of 3 elements. 1. RBS: BBa_B0034, upsteam one is reversed 2. Promoter: BBa_J23119, Bacterial constitutive promoter 3. att site: Recognition site for Mycobacteriophage Bxb1 integrase/excisionase We designed this part to generate two different proteins one at a time. At the first state, this device promotes transcription and translation of downstream gene because its promoter orientation. When Mycobacteriophage Bxb1 integrase recognizes and inverts sequence flanking with attB and attP sequence, promoter orientation reversed. Then this device promotes transcription and translation of upstream gene(must be reversed at the construction step). This device is tested by GFP and mRFP attached construct. See BBa_K090700N and BBa_K090700N
Promoter and RBS are derived from BBa_J23119 and BBa_B0034, respectively. Whole construct was de novo synthesized by Bioneer.
Artificial terminator, estimated %T~>90% * 8bp stem, 6nt loop * Bidirectional, estimated reverse %T~>90%
Bidirectional, with the reverse estimated to be more effective than the forward. Has a polyA tail of 4 residues
modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pairs
This is the pBAD promoter (BBa_I13453) in the opposite orientation. It can be used to drive transcription in the direction of suffix to prefix.
Rock - Primary plasmid (part 1) [RhlR generator]
Part of the iGEM2009 Team:Southampton rock-paper-scissors 'rock' primary plasmid.
Constitutive promoter selected to allow for continuous expression of C0171.
See relevant basic biobrick parts (J23119, B0034, C0171, B0015)
we synthesize the OR promoter by replacing OR3 binding site of lambda CI with OR1 binding site of CI434.
making sure CI434 protein can repress CI's transcription.
similar to lambda lysis/lysogenic regulatory region.
Double terminator consisting of BBa_B0012 and BBa_B0011
Edited Xylose Regulated Bi-Directional Operator 2
This part contains the operator region controlling transcription for the xyl operon. This regulatory region is dependent upon the binding of CRP-cAMP and XylR bound to xylose for activation. Upon binding, transcription is activated in both directions (xylFGH and xylAB facing). This part has a scrambled CRP binding site and and altered RNA ploymerase binding site that was designed to more resemble the consensus sequence at an intermediate level.
Both directions were kept in the sequence of the part even though it is intended to work in the right facing direction because of possible looping that may occur as a transcriptional control. The RNA polymerase binding site was altered to make binding stronger, attempting to allow activation when XylR-xylose alone is bound (the absence of CRP-cAMP).
Taken from E. Coli genome bases 3728830-3729092, base pairs changed at RNA polymerase bining sites.
Promoter from the lack operon cloned in the reverse orientation.
Scissors - Primary plasmid (part 2) [tac pR, mBanana and LuxI generator]
Part of the iGEM2009 Team:Southampton rock-paper-scissors 'scissors' primary plasmid.
Regulated expression of LasI (C0178) and mBanana (K180009) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, K180009, B0015).
Two T7 promoters are reversely overlapping in five base pairs, with the third one mutated from A to G.
The sequence of T7 promoter is TAATACGACTCACTATA (from 5' to 3').
Zymomonas mobilis glucose facilitator gene
Paper - Primary plasmid (part 2) [tac pR, GFP and RhlI generator]
Regulated expression of RhlI (C0170) and GFP (E0040) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, E0040, B0015).
Game of Life - Primary plasmid (part 2) [lux pR, GFP and LacI generator]
Part of the iGEM2009 Team:Southampton Game of Life primary plasmid.
Regulated expression of LacI (C0012) and GFP (E0040) by PLUX promoter (R0062), controlled by presence/absence of AHL-LuxR.
See relevant basic biobrick parts (R0062, C0012, B0034, K180001, B0015)
Bi-directional Terminator derived from pBR322.
This is an exact 'upside down' version of BBa_R0051. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. By adding this part, we wish to emphasise the possibility of engineering both DNA strands, and the additional regulatory potential that it gives.
Any 'upside down' (reverse complementary) sequences made by PCR amplification need be made with suffix and prefix is opposite order with respect to the original default sequence.
Rock-paper-scissors - Rock primary plasmid
Composite part, consisting of BBa_K180011 and BBa_K180012, but with BBa_K180011 reading along reverse strand, and BBa_K180012 reading along the forward strand. Forms the 'rock' primary plasmid in Team:Southampton's rock-paper-scissors project design.
This part has been specified as a basic BioBrick, due to some of the component parts reading in reverse (unable to specify using the component BioBrick format).
Artificial terminator, estimated %T~=80% # 8bp stem, 6nt loop # Bidirectional, estimated reverse %T~>90%
Bidirectional, with the reverse estimated to be more effective than the forward. Has a polyA tail of 6 residues.
Yan GONG, Shuobin CHEN, Yunpeng LIU, Xiaowei YAN
RFP+RBS_SDA-C1+OR21 double directed promoter+SDcro CI434+eGFP
mutate the RBS seqence change the translation rate of CI434 to get the appropriate ratio of green and red.
The part is made up of the standard parts from partsregistry, and some of the sub-parts are inverted by PCR.
Artificial terminator, estimated %T~=70% *8bp stem, 6nt loop *Bidirectional, estimated reverse %T~=80%
Bidirectional, with the reverse estimated to be more effective than the forward. Has a polyA tail of 5 residues.
modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pair
Rock - Primary plasmid (part 2) [tac pR, mCherry and LasI generator]
Regulated expression of LasI (C0178) and mCherry (K180008) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, K180008, B0015)
Two T7 promoters are reversely overlapping in two base pairs while a point mutation occurs at the 12th base pair, from A-T to T-A.
==Part Description== This is a composite part made from Green Fluorescent Protein (GFP) and a double terminator. Green Fluorescent protein (originally from part [http://partsregistry.org/Part:BBa_E0040 BBa_E0040]) that will glow green when excited by the correct wavelengths and thus can be used as a reporter gene. This part is directly fused to a terminator (originally from part [http://partsregistry.org/Part:BBa_B0014 BBa_B0014]) so it is ideal to be fused at the end of your construct. ==Table of Statistics== {| style="color:white; background-color:#0066CC;" cellpadding="20" cellspacing="0" border="2" !Chassis !Monomer/Multimer !Aminoacid Length/Weight !Localisation !Structure !Excitation wavelengths !Emmision wavelengths |- |Prokaryotic and Eukaryotic |Bipartite |274 aminoacids | |beta-barrel(for GFP) hairpins for Terminator |501nm |511nm |} ==References== <biblio> #Valdivia pmid=8707053 </biblio>
No additional design considerations. The parts were fused together following RFC10 assembly.
GFP originates from jellyfish Aequeora victoria. For more details visit the [http://www.expasy.ch/uniprot/P42212 SwissProt entry]. The double terminator was designed by Reshma Shetty. For more details visis the [http://partsregistry.org/Part:BBa_B0014 original part page].
Edited Xylose Regulated Bi-Directional Operator 1
This part contains the operator region controlling transcription for the xyl operon. This regulatory region is dependent upon the binding of CRP-cAMP and XylR bound to xylose for activation. Upon binding, transcription is activated in both directions (xylFGH and xylAB facing). This part has a scrambled CRP binding site and and altered RNA ploymerase binding site that was designed to more resemble the consensus sequence slightly.
Natural Xylose Regulated Bi-Directional Operator
This part contains the operator region controlling transcription for the xyl operon. This regulatory region is dependent upon the binding of CRP-cAMP and XylR bound to xylose for activation. Upon binding, transcription is activated in both directions (xylFGH and xylAB facing). This part has an unscrambled CRP binding site and and unaltered promoter region (unlike BBa_K100001 through BBa_K100003).
Both directions were kept in the sequence of the part even though it is intended to work in the right facing direction because of possible looping that may occur as a transcriptional control.
Taken from E. Coli genome bases 3728830-3729092
Bi-directional Terminator from pSMART HCKAN. E.coli derived, genbank U24206 bases 2694-2727 between yciA and tonA genes (converging transcripts)