1. Engineering the Subtilisin Gene into E. coli
MoldBusters
Oggie Golub
Katy Hood
Derek Stewart
Casy Cory
Josh Mauldin

2. Proposal Project Goal Vector Components
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)

3. Procedural Overview BioBrick Library Bacillus Subtilis
Extraction from Library
Extraction of Genome
Transformation
Amplification of Gene
Extraction of Plasmid
Digestion of Gene
Digestion of Plasmid
Prepared Plasmid
Prepared Gene
Ligation of Gene and Plasmid
nprE Gene
Wintergreen
Transformation into E. coli
Sequencing and Testing for Expression

4. Biobricks

5. 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

6. 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

7. 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

8. 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

9. Globiformis and Niger

10. 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

11. 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
1500
850
400
200 50

12. 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 + -
1500
850
400
200 50

13. 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

14. 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

15. Optimal Annealing Temperature Test
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.

16. 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

17. DNA Extraction Repeat Extract New DNA Agarose Gel Run
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

18. 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.

19. 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

20. 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
Ordered original strain from site

21. 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

22. 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.

23. Neutral Protease nprE

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. 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

32. Wintergreen

33. 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_
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).

34. Results: We had a misshapen gel, and our results were unreadable
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)

35. A Shift in gears: Wintergreen
The part was BBA_ , 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?

36. 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)

37. Results No bands, shadows may be gen. DNA.
Positive control and L.R.L in same well.
3/24/2009 (pg.27)

38. Re-Amplification Same protocol as before.
Hoping to eliminate and unknown errors in gel pouring, pipetting, ext.

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

40. 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.

41. 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
(pg.31) Amp of Wintergreen three two primer concentrations, and 2 DNA’s.

42. Mass wintergreen Production for ligation
Identical parameters for the band on previous gel in greater numbers.

43. 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
(See Katy Hoods notebook) Wintergreen Amp.

44. Wintergreen amplification …Thrid times the charm.
Amplify 10 samples of wintergreen.
Positive & Negative controls.
1/10 DNA dilution.
PCR Protocol “Katy”

45. Results: No amplification of wintergreen or positive control.
Realized we have been using 10X too concentrated primers.
Redo experiment.

46. FINAL Wintergreen amp. Using the proper concentration of primer.
DNA #7
Positive control PSB_1A7 (VF/VR)
Luck

47. Here we see a faint band around 1. 5 k. b
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.
(pg. 39) “Mega Final Wintergreen Amp.

48. 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.

49. Ligations, Transformations, and Results

50. Initial Primer Problems and Redesign
The proposed primer sequences were run through the IDT website. The following hairpins formed.
Forward Primer
Reverse Primer

51. 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

52. 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

53. 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) = ng/µL µL (volume of DNA loaded)

54. 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
Plasmid
8.0 µL
4.0 µL
Gene
0.1 µL
0.5 µL
10× Buffer
1.0 µL
DNA Ligase
Water
3.5 µL
7.5 µL

55. 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

56. 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

57. 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.