1. The Effect of IPTG Levels on Expression of the Lac Operon Joel Bradshaw W.F. West High School

2. Gene Regulation: The Basics Controls the production of proteinsControls the production of proteins Can be controlled by the presence of certain chemicals, or can be self- regulatingCan be controlled by the presence of certain chemicals, or can be self- regulating Examples: ROP, Lac OperonExamples: ROP, Lac Operon

3. Lac Operon: The Problem E. coli prefers GlucoseE. coli prefers Glucose Other energy sources availableOther energy sources available Must be digestedMust be digested Energy wasteEnergy waste HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH

4. Lac Operon: The Solution β-Galactosidase (Z) breaks down lactoseβ-Galactosidase (Z) breaks down lactose More β-Galactosidase neededMore β-Galactosidase needed Only when Glucose is not availableOnly when Glucose is not available HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH

5. Lac Operon: The Problem The Lac Operon is a set of genes and DNA regions that control the production of a proteinThe Lac Operon is a set of genes and DNA regions that control the production of a protein It is controlled by the presence or absence of lactose and glucose in the environmentIt is controlled by the presence or absence of lactose and glucose in the environment

6. The Lac Operon – How it works Three main regionsThree main regions –LacI site produces the repressor –CAP site produces the Catabolite Activator Protein –LacZYA region produces the protein and contains binding sites HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH

7. HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH Negative Control LacI Region produces repressorLacI Region produces repressor Repressor binds to operatorRepressor binds to operator When polymerase attaches to promoter, repressor stops transcriptionWhen polymerase attaches to promoter, repressor stops transcription

8. HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH Negative Control β-Galactosidase digests lactoseβ-Galactosidase digests lactose Break down of lactose causes allolactoseBreak down of lactose causes allolactose Allolactose releases repressorAllolactose releases repressor More lactose broken downMore lactose broken down NOT SO MUCH DETAIL! LacZ is replaced with GFP! – move to questions

9. Positive Control Cyclic AMP binds to CAPCyclic AMP binds to CAP –Compound helps bind polymerase to promoter Glucose inhibits formation of cyclic AMPGlucose inhibits formation of cyclic AMP Polymerase achieves little transcriptionPolymerase achieves little transcription HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH NO HIGH LEVELS TWICE!

10. Positive Control Glucose levels are low - cyclic AMP can formGlucose levels are low - cyclic AMP can form Cyclic AMP binds to the CAPCyclic AMP binds to the CAP –Helps polymerase bind to promoter Polymerase can transcribePolymerase can transcribe HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH

11. How it applies IPTG imitates allolactoseIPTG imitates allolactose GFP inserted into LacZYAGFP inserted into LacZYA HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH

12. ROP: The Problem

13. ROP: The solution The ROP region controls the copying of the plasmidThe ROP region controls the copying of the plasmid Ensures a low copy numberEnsures a low copy number Eliminates the variable number of plasmids in a bacteria, ensuring regular measurementsEliminates the variable number of plasmids in a bacteria, ensuring regular measurements

14. The ROP protein, increases the affinity of the primers for each other, so they don’t stick to the Origin of Replication, causing the plasmid to cease replicating.The ROP protein, increases the affinity of the primers for each other, so they don’t stick to the Origin of Replication, causing the plasmid to cease replicating. ROP ORI ROP: How it works ROP ORI

15. Hypothesis Increased IPTG concentration will result in increased expression of the Lac Operon

16. Questions Effect of IPTG concentration on lac expressionEffect of IPTG concentration on lac expression –Does IPTG affect lac expression? –How does it affect expression? –Is there a limit to how much IPTG affects?

17. Experimental Design Engineer a plasmid with the ROP and the Lac OperonEngineer a plasmid with the ROP and the Lac Operon Grow the plasmid in varying concentrations of IPTGGrow the plasmid in varying concentrations of IPTG Measure the amount of GFP productionMeasure the amount of GFP production

18. Creating the pNGN pUC19 pBR322 GFP

19. Creating the pNGN Amplified desired sectionsAmplified desired sections Restricted pBR & pUCRestricted pBR & pUC GFPpBRpUC 2612 bp 23130 9416 6557 4361 2322 2027 564 707 bp

20. Ligated pBR and pUCLigated pBR and pUC Transformed ligations into E. coliTransformed ligations into E. coli Spread on plates with ampicillin to select for bacteria with pUC19Spread on plates with ampicillin to select for bacteria with pUC19 Creating the pNGN Marker 10 ul 30 ul pBR Rest. pUC Rest.

21. Creating the pNGN Plucked colonies from plates and grew in OSC’sPlucked colonies from plates and grew in OSC’s Extracted, restricted to separate targeted sectionsExtracted, restricted to separate targeted sections #5 has pUC and pBR fragments, and nothing else#5 has pUC and pBR fragments, and nothing else Marker #1 #2 #3 #4 #5 Marker 23130 9416 6557 4361 2322 2027 564 2612 bp 707 bp

22. Creating the pNGN Ligated #5 and GFP fragmentsLigated #5 and GFP fragments Transformed ligations into E. coliTransformed ligations into E. coli Spread on platesSpread on plates –Ampicillin (pUC) –IPTG (GFP) Selected 10 glowing coloniesSelected 10 glowing colonies Grew in OSC’sGrew in OSC’s

23. Creating the pNGN Extracted DNA from OSC’s and restricted to separate fragmentsExtracted DNA from OSC’s and restricted to separate fragments Selected #6 due to concentrationSelected #6 due to concentration 100 bp Marker #1 #2 #3 #4 #5 Marker #6 #7 #8 #9 #10 100 bp Marker

24. Creating the pNGN Sent pNGN to be sequencedSent pNGN to be sequenced Both ROP & GFP verifiedBoth ROP & GFP verified

25. Experimental Design Experimental groupsExperimental groups –Negative Control E. coli with no plasmidE. coli with no plasmid E. coli with no plasmid + 10µl IPTGE. coli with no plasmid + 10µl IPTG –IPTG levels (µl per 5 ml broth) 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 200, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 Grew conicals for several hoursGrew conicals for several hours

26. Measuring Fluorescence Pelleted cellsPelleted cells Resuspended cellsResuspended cells Measured in fluorometerMeasured in fluorometer

27. Results As IPTG increased, fluorescence increased – to a pointAs IPTG increased, fluorescence increased – to a point Once IPTG reached a threshold, fluorescence dropped off and slowly decreasedOnce IPTG reached a threshold, fluorescence dropped off and slowly decreased

28. Results Fluctuations, but still peaked near 10Fluctuations, but still peaked near 10 Increase after 18Increase after 18

29. Results

30. Statistical Analysis Fluorescence Time0102030E. coliCorrelation 2:002319171828-0.834 2:3028374028240.063 3:0022384049280.954 3:3026455163340.979 4:1529566059400.822 5:053398122102410.772 5:4555188182173460.710 15:212565407101296137.968 59.000127.625152.750223.50047.2500.986 P- Value0.2420.1290.0930.084

31. Discussion Increased IPTG = increased GFP (LacZYA) productionIncreased IPTG = increased GFP (LacZYA) production Expression reduced at high levelsExpression reduced at high levels –Loss of function due to wasted energy –Reduced GFP production

32. Limitations Trials 1 & 2Trials 1 & 2 –Amount of time grown Trial 3Trial 3 –Amount of initial broth –Length of measurement period

33. Future Research Compare IPTG to LactoseCompare IPTG to Lactose Test effects of glucoseTest effects of glucose Test farther out to investigate the bump in second trialTest farther out to investigate the bump in second trial Control time grownControl time grown

34. Acknowledgements Mr. Henri Weeks, W.F. West High SchoolMr. Henri Weeks, W.F. West High School My Family, for tolerating my long absencesMy Family, for tolerating my long absences The FedEx lady, for waiting to take our samples for sequencingThe FedEx lady, for waiting to take our samples for sequencing

35. Questions?

36. pUC19

39. pBR322

43. pGLO

44. Misc CorrelationCorrelation –Upward: 0.942507 –Downward: 0.87545 –Curve: R 2 =.8478 IPTG: Isopropylthiogalacto pyranosideIPTG: Isopropylthiogalacto pyranoside

45. HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH Purpose of the Lac Operon The genes in ZYA digest lactoseThe genes in ZYA digest lactose –Z = β-Galactosidase (breaks down lactose) GlucoseGlucose GalactoseGalactose –Y = Lactose Permease (transports lactose into cell) –A = Thiogalactoside transacetylase (toxicity) A side effect of the break down of lactose is allolactoseA side effect of the break down of lactose is allolactose Allolactose binds to repressor and relelases itAllolactose binds to repressor and relelases it NOT SO MUCH DETAIL! LacZ is replaced with GFP! – move to questions

46. Results 0102030E. coli0102030E. coli 8:00 23191718280.0740.0720.0730.0690.082 8:30 28374028240.0720.0810.0860.0780.074 9:00 22384049280.1240.1310.1250.121 9:30 26455163340.1510.1630.1660.1540.146 10:15 29566059400.2170.2200.2350.2140.199 11:05 3398122102410.3030.3220.3180.3040.314 11:45 55188182173460.4230.4350.4360.4230.410 21:21 25654071012961370.8820.7720.8050.8660.970 0.940390.964320.954190.936010.97552 Fluorescence E. Coli density