SYNCHRONIZED, CONDITIONAL, GENETIC CHEMOTAXIS PROGRAMMING
From left to right: Mr. Gert Pietersen, Dr. Eshchar Mizrachi, Ms. Modjadji Makwela, Mr. Axel Ind, Ms. Thabang Msimango, Mr. Vaughn Barendsen, Ms.
Nomakula Zim, Mr. Ricu Claassens, Dr. Steven Hussey, Prof. Zander Myburg and Mr. Brad Querl
Seven undergraduate students and one BSc. Honoursstudent were selected for team Pretoria_UP, the first iGEMteam at the University of Pretoria. Our subject areas rangefrom human genetics to plant science, microbiology tocomputer science. We are instructed by Dr. Steven Hussey,advised by Prof. Zander Myburg and Dr. Eshchar Mizrachi,and hosted in the Forest Molecular Genetics Programme.
Six synthetic DNA constructs were ordered from Integrated DNA Technologies (IDT).Parts BBa_K1768000 through BBa_K1768006 were documented on the iGEM Registry.
CLONING: gBlock parts cloning from a restriction digest (Left) and PCR amplification of the Biobricks (recombinase switch module) corresponding to their expected sizes (right).
SEQUENCING: When analysing the sequence datait was found that the Cre generator constructcontained two large deletions in the coding regionof the construct. The seven bp deletion causesframe shift and would render the recombinaseprotein deactivated. The other gBlock fragmentsequences were correct with no mutations. Wedid not have sufficient time to re-order or reclonethe Cre generator, but this is under way.
We would like to convey our gratitude to the following individuals who helped make our project a success: Prof.Jacques Theron, Marilyn Ekoka, Dr. Marco Weinberg, Natascha Muller, Schae Ind, Julie Ind, Elodie Ekoka, DanielleRoodt, Colin Balkwil, Drew Behrens and Jonathan Botha.
South Africa has a turbulent history of racial segregation and its impact can stillbe seen today in education. Due to the contrasting socio-economic circumstanceswhich still prevail, we realised that learners from previously disadvantaged areasmay not have been exposed to the promise of synthetic biology (SynBio). Weissued surveys to Grade 11 learners in two schools situated in different socio-economic areas, Lehlabile Secondary School (LSS) and Pretoria Boys High School(PBHS).
POSITIVE CONTROLS: RFP (left top, BBa_I13522) and GFP (leftbottom, BBa_I13521) positive control constructs performed asexpected (100% of cells expressed the marker).
CONCLUSIONS AND FUTURE WORK: Our characterisation workwas interrupted due to a problem in the Cre recombinaseconstruct. The Cre recombinase part (BBa_K1768004) will haveto be re-ordered. Cre-mediated recombinase switches will betested after IPTG induction of Cre using fluorescent microscopy(% RFP-positive cells) and DNA sequencing of RFP-expressingcolonies. A His tag was fused to the Cre protein to monitor itsexpression via Western Blot analysis.
INVERTER SWITCH: By flanking a constitutivepromoter with recombinase recognitionsequences, a stable change of gene expression(GFP to RFP reporter genes) is induced throughinversion of the DNA (promoter). The lox66 andlox71 elements are recognised by the Creprotein which facilitates recombination andthus triggers a non-reversible change in geneexpression. RFP is on the antisense strand.
• Quorum sensing is employed to ensure the bacteria behave as aswarm and not individual units.
• A theophylline riboswitch is used to perceive when the bacteriacross a concentration gradient threshold.
• AND gate logic is used to activate a gene switch when input fromquorum AND the theophylline riboswitch modules are received.
• Chemotaxis is controlled by a recombinase gene switch under thecontrol of the AND gate. This module induces a change indirection of chemotaxis.
• A reporter module relays information about a substance ofinterest.
CHEMOTAXIS: The ability to control bacterial motility is importantto synthetic biology due to the variety of potential applicationssuch as the “Biotweet” project of the WITS-CSIR_SA team of 2011.
LIMITATIONS: Some of the shortcomings of the “Biotweet” projectdesign is the asynchronous behaviour of the bacteria resulting inreversal of direction before reaching the “recipient” position.Further, the original design does not communicate any informationin “reply” from the recipient.
IMPROVED DESIGN: The Pretoria_UP team proposes designs for asynchronous “reply-tweet” that is conditional on a large enoughquorum presence and are able to detect and communicateinformation from the recipient such as whether a chemical ofinterest is present.
CHARACTERISATION: The genetic switch which would inducechemotactic reversal in the original designs have not beencharacterised. It is our aim to characterise several designs for LoxP-Cre recombinase-based genetic switches.
Gert Pietersen1,3, Nomakula Zim3, Vaughn Barendsen3, Ricu Claassens3, Axel Ind3, Modjadji Makwela3, Thabang Msimango3, Brad Querl3, Eshchar Mizrachi2,3, Zander Myburg2,3 and Steven Hussey2,3
1Department of Microbiology, 2Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), 3University of Pretoria, Private Bag X20, Pretoria 0028, South Africa
EXCISION SWITCH: A transcriptionaltermination sequence (after the GFP reporter)insulates RFP from being activated by aconstitutive promoter. Cre-mediated excision ofthe terminator via lox66 and lox71recombination sites results in activation of RFPand loss of GFP.
RECOMBINASE GENETIC SWITCH: AND gate outputinduces recombination and downstream “reply-tweet”
Educational background:Grade 11 learners from LSSand PBHS schools showedsignificant differences in agedistribution, family educationand previous exposure toSynBio (P < 0.05)
Acceptance: LSS was moreaffirming than PBHS, but bothschools responded positivelytoward synthetic biologyproduct consumption
Future vision: LSSviewed SynBio asapplicable to primaryneeds, whereas PBHSlearners were morenegative aboutSynBio applications(P < 0.05).
Impact: LSS participants were clearly excited about the future prospects of SynBio. A positive but somewhatmore sceptical response was observed for PBHS learners, a significant difference between the two schools.
Conclusion: Previously disadvantaged (LSS) andadvantaged (PBHS) learners showed significantdifferences in SynBio awareness, acceptance andexpectations. The latter were more sceptical anddistrusting of this field, whereas the formerregarded it as a means to providing primaryneeds.
Funding bodies:• Department of Science and
Technology• Foresty Molecular Genetics
group• Sappi Ltd• Mondi Ltd
Instructors and advisors:• Dr. Steven Hussey• Prof. Zander Myburg• Dr. Eshchar Mizrachi
