Engineering the Subtilisin Gene into E. coli

MoldBustersOggie GolubKaty Hood

Derek StewartCasy Cory

Josh Mauldin

Proposal

• Project Goal– To engineer the aprE gene into E. coli and test for

expression of subtilisin.

• Vector Components– Antibiotic Resistance Plasmid pSB1A2 (or pSB1A7)

– Arabinose Induced Promoter R0080 (or I0500)

Procedural OverviewBioBrick LibraryBioBrick Library Bacillus SubtilisBacillus Subtilis

Extraction from Extraction from LibraryLibrary

TransformationTransformation

Extraction of Extraction of PlasmidPlasmid

Digestion of Digestion of PlasmidPlasmid

Extraction of Extraction of GenomeGenome

Amplification of Amplification of GeneGene

Digestion of GeneDigestion of Gene

Ligation of Gene Ligation of Gene and Plasmidand Plasmid

Transformation into Transformation into E. coliE. coli

Sequencing and Sequencing and Testing for ExpressionTesting for Expression

Prepared PlasmidPrepared Plasmid Prepared GenePrepared Gene

WintergreenWintergreennprE GenenprE Gene

Biobricks

Cloning Plasmid

• Extracted part pSB1A2+R0080 and pSB2K3+I0500– Extracted from 2007 & 2008 Library

– Followed Protocols from both Libraries

• Transformed into Competent Cells– Plated transformants

• pSB1A2+R0080 on Ampicillin

• pSB2K3+I0500 on Kanamycin

– Results

• Only Cells with pSB1A2+R008 from 2007 Showed Growth

• Prepared Glycerol Stocks and Backup Plates

Extracting Plasmid

• Extracted Plasmid from Transformed Colonies– Followed Protocol from GeneJET Mini Prep Kit

• Ran Digested and Undigested Plasmid on a Gel– Digested Samples Contained Plasmid and Promoter

Fragments

Preparing Plasmid for Ligation

• Digested Plasmid– Used SpeI and PstI Restriction Enzymes

– Followed Protocol from QIAquick PCR Purification Kit

• Ran Digested Plasmid on Gel– Both the Digested Plasmid and Undigested Plasmid were

present

Preparing Plasmid for Ligation

• Extracted Digested Plasmid from Gel– Top Band was Excised from the Gel

– Followed Protocol from QIAquick Gel Extraction Kit

• Ran Gel to Confirm the Plasmid was Ready for Ligation

Globiformis and Niger

Glycerol Stock Preparation

• Glycerol Stocks Prepared– Four overnight cultures were obtained from Dr. Walter

(Niger and Globiformis strains)

• DNA Extraction of B. subtilis– We used both strains for our extraction

– The procedure we used was found online at OpenWetWare

DNA Extraction Results

• Agarose Gel Results– The results showed no DNA

present and we ran another gel to double check, which yielded about the same results.

– We decided to run a PCR on the DNA anyways because there was a possibility that something might be present

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PCR Amplification of aprE

• Used a standard PCR setup• Agarose Gel Run of PCR

– The positive control worked – The DNA did not show up

very bright– Possible problems:

• Incorrect Primers• Impure DNA• DNA Extraction Errors

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400

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+ -

Troubleshooting our DNA Extraction

• We wanted to find out if our DNA extraction procedure works for our DNA samples or if another procedure would allow us to obtain more purified DNA.

• We found a new DNA extraction method online and obtained new DNA to try out this new procedure

• Extraction protocol found on www.Bio.net

Agarose Gel Run of New DNA

• The results were unclear for this particular gel because the gel had been sitting too long before we loaded our samples

• The result was that everything was blurry and fuzzy

• We believe, however, that some DNA was present

Optimal Annealing Temperature Test

• Annealing Temperature– We ran a gradient at five temperatures to find an optimal

annealing point for our future PCR

• 48°, 50.1°, 52°, 54.7°, and 56.8°

– We made five globiformis mixes, five niger mixes, a positive control, and a negative control for this PCR.

Results

• Agarose Gel Run– Our results did not turn out

well

– The Niger samples were very bright

– Neither the Niger or the Globiformis samples appeared to have much DNA present

DNA Extraction Repeat

• Extract New DNA– We extracted new DNA from

both variations to use when we’re cloning the aprE gene

– We used the same protocol as last time

• Agarose Gel Run– We finally had a little success

this time and were able to see that DNA was present

Annealing Temp Test #2

• Ran an annealing gradient for PCR to find optimal annealing point

– We used the same setup and temperatures as before

– The positive control didn’t show up.

– Since our controls did not work, there is no way to tell if the PCR is running properly.

Conclusion

• Since none of our PCR’s have been successful we need to come up with backup plans– We are going to try using a plasmid that was made last

year by a previous group

– We are also going to research obtaining new primers since we are not having any success with our present ones

Locating Strain 168

• We researched the Bacillus Subtilis Strain 168 online to try and find somewhere we could purchase it from

• Bacillus Genetic Stock Center• www.bgsc.org

– Ordered original strain from site

Reviving B. subtilis 168

• Revive Bacillus Cultures (Strain 168)– Arrived in the mail as spore dots on filter disks– Used the instructions that came with the cultures to revive them– The bacteria colonies revived well– We made two liquid cultures and streaked two on LB plates– Josh and Casy used these cultures to do a DNA extraction and

continue on the Bacillus subtilis route while Derek and I started a backup plan using Wintergreen

– Oggie worked on cloning the nprE gene

Extraction of Genomic DNA

• Extracted Genomic DNA from B. subtilis 168– Followed Protocol Found on BioNet

– Digested and Undigested Samples were Run on a Gel

• The Digested Samples were Digested with EcoRI.

Neutral Protease nprE

Neutral Protease (nprE) Project Design

• Aside from subtilisin (aprE) B. subtilis also produces a neutral protease called bacillolysin– Coded for by the nprE gene

– Has properties similar to subtilisin

• Alkaline protease vs. neutral protease

• Used NCBI genome database to design forward and reverse primers

Primer Design

• Three forward and one reverse primer was used to clone the nprE gene– F1 designed for entire nprE gene

– F2 designed for just the mature product

– F3 was a mutated version of F2

• Contained less hairpins

– All three contained the same prefix

– Reverse contained the same suffix

Preparing Primers

• Primers came not dissolved– Dissolved in U.V. treated water to 100 μM

• After dissolving they needed to be dilluted– 100 μM to 10 μM dillution using U.V. treated water

• Setup a PCR gradient to determine optimal annealing temperatures

Annealing Temperature Test Results

• Failure again..– + control worked

– No 1500 bp or 900 bp bands present

• “Mystery” band around 200 bp instead

– - controls showed nothing

• All results look the same– Primers self annealing?

– Not according to negative controls

Testing DNA & R1 Primer

• “Mystery” band of 200 bp showed up only in the presence of DNA and R1 primer– Reverse primer acting as forward as well?

– DNA has fragments which act as primers?

• Tested by running PCR with only DNA, and only the reverse primer

Results

• “Mystery” band showed up with just DNA– Band must be part of

genomic DNA

• Reverse primer only showed nothing– Not acting as forward

primer

Troubleshooting previous aprE failures

• Josh & Casy succeeded in cloning the aprE gene– Modified primers and used new strain

• Which one resulted in success?

• Performed tests – Old strains with newly modified primers

– New strain with original primers

Results

• It was necessary to change both the DNA and primers– 168 w/ old primers

– G & N w/ new primers

• All look like negative controls

– - Controls

• Primers only

Wintergreen

Positive Control:Plasmid Test

• Had reoccurring problems with positive controls in our PCR reactions

• Wanted to test two different plasmids as our positive controls• We used P_Bluescript and BBA_1765001.• To assess why we were having bad results, we used two other

variables. Temperature (48-56 degrees) and primer dilution (1/100, 1/10 and 1/1).

Results: We had a misshapen gel, and our results were unreadable. Not shown are results from another group which confirm the PCR worked.

3/10/2009 (pg.23)

A Shift in gears: Wintergreen

• The part was BBA_1765001, which was already inside a plasmid.

• Used WGF1 and WRR2 primers.• Gene of about 1300 b.p. will be cloned from the

construct prepared last semester by “The sweet smell of, E.Coli?

Initial Amplification

• We were supplied with three samples of plasmid containing the wintergreen gene (#’s 3,7,8).

• Positive control psB_1A7 (confirmed to work by other group)

• Three annealing temperatures: 48, 52, 56 degrees.• PCR program “Onion 1” (See pg.21)

Results

• No bands, shadows may be gen. DNA.• Positive control and L.R.L in same well.

3/24/2009 (pg.27)

Re-Amplification

• Same protocol as before.• Hoping to eliminate and unknown errors in gel

pouring, pipetting, ext.

Results:No banding. However, DNA #3 showed little to no coloration.Proceeded using DNA #7 and #8 due to activity shown.

Wintergreen Amplification with additional primer.

• Attempt to:• Amplify the Wintergreen using both DNA #7 & 8.• Three temperatures as before.• Primer concentrations of 1/1 and 1/10.• Test integrity of plasmid using VF and VR.

• Unfortunately, ran the agarose gel too long, and samples ran into each other. However, we can see a clear band at the appropriate size for wintergreen. Also, all VF/VR samples amplified regardless of temperature.

• The wintergreen

only amplified

at 48 degrees.

The band we see

Had a 1/10 primer

Dilution. DNA #7

3-4-09 (pg.31) Amp of Wintergreen gene @ three temps. @ two primer concentrations, and 2 DNA’s.

Mass wintergreen Production for ligation

• Identical parameters for the band on previous gel in greater numbers.

• No amplification of the Wintergreen gene. • Due to some confusion, we had three positive controls, and

three negative controls – all had expected results.• We conclude we have misinterpreted our results from 3-4-09.

4-9-09 (See Katy Hoods notebook) Wintergreen Amp.

Wintergreen amplification …Thrid times the charm.

• Amplify 10 samples of wintergreen.• Positive & Negative controls.• 1/10 DNA dilution.• PCR Protocol “Katy”

Results:• No amplification of wintergreen or positive control.• Realized we have been using 10X too concentrated

primers. • Redo experiment.

FINAL Wintergreen amp.

• Using the proper concentration of primer.• DNA #7• Positive control PSB_1A7 (VF/VR)• Luck

• Here we see a faint band around 1.5 k.b. This is in the expected range of the Wintergreen gene. Our positive and negative controls worked properly.

• You will see two positive controls, one has .8ul VF/VF primer and one has 1.0ul. As you can see, there was no appreciable difference in the two.

4-21-09 (pg. 39) “Mega Final Wintergreen Amp.

Final Thoughts..

• We had several mistakes…Wrong primer concentration, misinterpreted results,

double well loading, wrong primer, mass confusion. The wintergreen yielded little to (probably) no actual

gene amplification.

Still loved the class.

Ligations, Transformations, and Results

Initial Primer Problems and Redesign

• The proposed primer sequences were run through the IDT website. The following hairpins formed.

Forward Primer Reverse Primer

Amplification of the aprE Gene

• PCR Setup– Temperatures of 45°C, 50 °C, 55 °C, and 60 °C

– Template Volumes of 0.1 µL and 1 µL

• Ran Gel of PCR Products– Verified aprE Gene was Amplified

Preparing aprE Gene for Ligation

• Repeated PCR• Digested aprE Gene

– Used XbaI and PstI Restriction Enzymes

• Ran Gel of Digested Gene– Top Band was Excised and Extracted

Preparing aprE Gene for Ligation

• Ran Gel to Estimate Gene Concentration– Concentrated and Dilute Samples were Loaded

– Concentration Calculated to be 13.3 ng/µL

40 ng/10 µL (determine by ladder) = 13.3 ng/µL 3 µL (volume of DNA loaded)

Ligation of the Gene and Plasmid

• The Digested Gene and Plasmid were used for the Following Ligations

Molar Ratio (Plasmid:Gene)

2:1 1:1 1:5 Control Control

Plasmid 8.0 µL 4.0 µL 4.0 µL 0 4.0 µL

Gene 0.1 µL 0.1 µL 0.5 µL 0.5 µL 0

10× Buffer 1.0 µL 1.0 µL 1.0 µL 1.0 µL 1.0 µL

DNA Ligase 1.0 µL 1.0 µL 1.0 µL 1.0 µL 1.0 µL

Water 0 4.0 µL 3.5 µL 7.5 µL 4.0 µL

Cloning Ligated Plasmid

• Transformed Ligation Products into Competent Cells– Selected for Ampicillin Resistance

– Results

• Majority of Colonies Resulted from the Ligation with the 1:1 Molar Ratio

• Ligation Appeared Successful

Extracting Ligated Plasmid

• Extracted Plasmid from Transformed Colonies– Followed Protocol from GeneJET Mini Prep Kit

– Sequenced Samples

• Ran Digested Samples on a Gel– Digested with XbaI and SpeI

– Expected Two Fragments

• Promoter–Gene at 1300 bp

• Plasmid at 2000 bp

Project Results

• The aprE gene was not present the colonies obtained.• Sequencing revealed the plasmid as expected, as well

as the promoter flanked by the appropriate BioBrick prefix and suffix.

• Possible explanations:– Gene and Plasmid did not properly ligate.

– Transformation was inefficient.

– Promoter was constitutive resulting in immediate cell death.