BioSketch The bacterial sketch pad.

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

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. 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) • lcI-only test construct (pWCI) • lacI-only test construct (pEL) • Other constructs

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

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

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. 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. 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. pWCI Ptrc l cI lcI-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

10. PL* lacI pEL 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

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. What has been done with Biobricks • Pairwise assembly: • QPI digestions successful with modified protocol: • Pl + QPILac • QPIl + YFP • QPI434 + 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 QPILacI(ts) mutants (m241 and m265) for sequencing • Outsourced 2 different ts, ind- versions of lcI. (“mJP3” based on Japanese patent Genbank #E07700, and “m8572” based on cI857 sequence found in HENDRIX Lambda II book).

13. Successful Ligations: mCherry + Terminator RBS + 434 RBS + lcI RBS + mCherry-Terminator RBS + Venus-Terminator Digestions ready for ligation: PlambdaCI P434 PlacI PlacI(hybrid) 434 lcI RBS-434 RBS-lcI RBS-mCherry-Terminator RBS-Venus-Terminator QPIlacI QPIl QPI434 Ligations

14. Summary: What we have done • CollinsMod: • Reconstituting the Collins circuit • The GFP reporter works • The P(L*) is constitutive in the absence of lcI • 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

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/m2 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. This week with Biobricks • More Pairwise Assembly • First Round • PlacI(hyb) + RBS-lCI • Pl + RBS-434 • RBS + LacIts(265) • All promoters + mCherry • All promoters + Venus • Second Round • Terminator + P434-mCherry • Terminator + P434-Venus • Terminator + Pl-mCherry • Terminator + Pl-Venus • Sequence cI857 mutations • Test microscopy with Promoter-Reporter constructs • Are mCherry-expressing colonies red enough? • Make bacterial mCherry into BioBrick • QPIl + mCherry • QPI434 + mCherry • Pl + QPIlacI • Pl-QPIlacI + QPIl-mCherry • Pl-QPIlacI + mCherry • PlacI + QPIl-mCherry • Pl + QPI434-mCherry