1. BioSketch The bacterial sketch pad. Yves Wang, Jennifer Gao, Yin Li Chris Doucette, Thomas Noriega, Hing Eng Group Meeting 2005-07-05

2. BioSketchHarvard iGEM 2005 2 Talk outline What has been done with the Collins constructs What has been done with the BioBricks What’s in store for this week

3. BioSketchHarvard iGEM 2005 3 The CollinsMod Team Reconstituting Collins Circuit Preliminary tests in TOP10 background Testing in the lacI- backgrounds Building the constructs Thermosensitive-LacI toggle-switch constructs (pTS241; pTS265) cI-only test construct (pWCI) lacI-only test construct (pEL) Other constructs

4. BioSketchHarvard iGEM 2005 4 Preliminary Tests: Procedure 1. Overnight culture in selective LB 2. Dilute and incubate areobically overnight in selective LB with either 0 or 2mM IPTG 3. Spin down and resuspend for concentrated cell suspensions 4. Examine under microscope

5. BioSketchHarvard iGEM 2005 5 Preliminary Tests: They Glow! pWG-only transformants w.o. IPTG 60x objective

6. BioSketchHarvard iGEM 2005 6 Preliminary Tests: Results Summary: No major difference between IPTG+ and IPTG- Much higher fluorescence in pWG (reporter) alone than in pWG+pTS Not highly instructive since the TOP10 genotype is very different from that of the strain Collins used Fluorescence is also seen on agar on petri plates, although stage not optimal MIT microscope for imaging fluorescence in worms might be more appropriate

7. BioSketchHarvard iGEM 2005 7 Reconstituting in lacI- backgrounds MC4100 from the Registry Competent MC4100 cells have been prepared Already transformed into cells: pWG pWG + pTS pWG + pTS241(11) pWG + pTS265(1) Ready to be tested JM 2.300 from Collins Has been requested

8. BioSketchHarvard iGEM 2005 8 Thermosensitive LacI Toggle Switches The toggle switch constructs w. the ts mutations Ala241->Thr: pTS241 Gly265->Asp: pTS265 The entire insert regions of two independent clones of both constructs have been sequenced Chose to use pTS241(11) and pTS265(1) Curious observation: Both ts sequences share three substitutions not expected from the sequence Collins gave us for the parental plasmid pTS Suggests that mutations are actually in pTS

9. BioSketchHarvard iGEM 2005 9 cI-only test construct cI-only test construct pWCI: Retransformation using the plasmid Collins sent us again gives restriction fragments identical to those of the toggle switch construct pTS pWCIb Strategy: delete the LacI region from pTS Results: analytical digets of five clones shows correct fragments; needs to be sequenced cI cI P trc pWCI

10. BioSketchHarvard iGEM 2005 10 lacI-only test construct pEL: Pursuing two different strategies: pELa: deleting the cI region from the toggle switch pTS pELc: excising the lacI and cI regions from pTS and re- ligating lacI back into the pTS vector Problems: pELa originally faced the problem of XbaI not cutting Suspected dam methylation pTS miniprepped from dam- strain still does not cut, however pELc requires Klenow rxn and CIPping No transformants so far PL*PL*PL*PL*lacIpEL

11. BioSketchHarvard iGEM 2005 11 Other Contructs Test reporter for CI regulation mCherry (pWC) Bacterial mCherry in pRSET-B vector arrived from MIT Waiting on the 70-mer PCR cloning primer Test reporters for LacI regulation GFP (pEG) mCherry (pEC) Cloning strategies have been devised; primers to be ordered Composite GFP/mCherry reporter for toggle switch pTSGC Waiting on pWC and pEG

12. BioSketchHarvard iGEM 2005 12 What has been done with Biobricks Pairwise assembly: QPI digestions successful with modified protocol: P + QPI Lac QPI + YFP QPI 434 + YFP Completed first two rounds and started third round of ligations to build QPIs from scratch Performing ligations in duplicate to decrease chances of setbacks Sequenced successful ts (mut265) mutations in LacI Have sent LacI ts (mut241) and both QPI LacI(ts) mutants (m241 and m265) for sequencing Outsourced 2 different ts, ind- versions of cI. (“mJP3” based on Japanese patent Genbank #E07700, and “m8572” based on cI857 sequence found in HENDRIX Lambda II book).

13. BioSketchHarvard iGEM 2005 13 Ligations Successful Ligations: mCherry + Terminator RBS + 434 RBS + cI RBS + mCherry-Terminator RBS + Venus-Terminator Digestions ready for ligation: P lambdaCI P 434 P lacI P lacI(hybrid) 434 cI RBS-434 RBS- cI RBS-mCherry-Terminator RBS-Venus-Terminator QPI lacI QPI QPI 434

14. BioSketchHarvard iGEM 2005 14 Summary: What we have done CollinsMod: Reconstituting the Collins circuit The GFP reporter works The P(L*) is constitutive in the absence of cI Building constructs Built pWCI Little progress on pEL and other constructs BioBricks: Pairwise assembly QPI digest problem solved; ligations still in testing Mutations and Sequencing We have a sequenced Bb-LacI m265 We are sequencing LacI m241 and QPI-lacI m241 and m265 Introducing mJP3 and m8572 into Lambda cI and QPI-Lambda cI mCherry + T RBS + lcI RBS + mCherry-T RBS + 434 RBS + Venus-T

15. BioSketchHarvard iGEM 2005 15 This week with CollinsMod Testing in lacI- strains Reproduce Collins' results: Incubate o.n. in 2mM IPTG Irradiate w. 0, 6, 12, 24 J/m 2 UV With o.n. in 2mM IPTG Examine what "initial state" is Examine how long initial state is maintained w.o. IPTG With o.n. @ 40C w.o. IPTG Examine if state is same by raising incubation temperature for the ts toggle-switches Building constructs Sequence pWCI Try to get pEL, along w. pEL241 and pEL265 Order primers for pEG and pEC

16. BioSketchHarvard iGEM 2005 16 This week with Biobricks More Pairwise Assembly First Round P lacI(hyb) + RBS- CI P + RBS-434 RBS + LacIts(265) All promoters + mCherry All promoters + Venus Second Round Terminator + P 434 -mCherry Terminator + P 434 -Venus Terminator + P -mCherry Terminator + P -Venus Sequence cI857 mutations Test microscopy with Promoter-Reporter constructs Are mCherry-expressing colonies red enough? Make bacterial mCherry into BioBrick QPI + mCherry QPI  + mCherry P + QPI lacI P -QPI lacI + QPI -mCherry P -QPI lacI + mCherry P lacI + QPI -mCherry P + QPI 434 -mCherry