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PROJECT LAB BOOK

Welcome to the Macquarie University project lab book page!

DAY-BY-DAY PROGRESS FOR THE TRANSFORMATION AND CLONING

22nd September 2010

Plasmid Prep using Qiagen Plasmid Midi Prep Kit

The pET-3A vector plasmid that is to be used for the transformation needs to be purified

The pET-3A plasmid was purified using the Qiagen Plasmid Midi Prep Kit as per the manufacturer’s protocols

The only difference made to the protocol is that only 5ml of Buffer QC was used on the 2nd wash step

The plasmid (two samples A & B) as well as some column flow-through collected at different stages of the purification were run on a GelRed post-stained 1.5% agarose gel and photo taken for visualization (see below).

A NanoDrop spectrophotometer reading was also recorded to check the quality of the extracted genomic DNA.

There was no DNA pellet formed at the end of this protocol so we may have lost some DNA

There was also some plasmid observed in some of the column flow through so some plasmid has been lost

Figure 10. Qiagen Plasmid Midi Prep results:

Figure 10. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid purification. In lanes 1, 4, 5 and 15 there is a 1kb Molecular ladder. In lane 2 there is the Plasmid purification sample A and in lane 3 is the Plasmid purification sample B. In lanes 6 to 14 there are flow-throughs that were collected at different stages throughout the protocol. There is some DNA visible in these lanes so some of the DNA has been lost. Possibly genomic DNA they are quite high (didn’t move through the wells)

Nanodrop results:

Sample	Concentration	A260/280 ratio
A	36.6ng/uL	1.71
B	35.0ng/uL	1.73

Looking at these results it seems that we have approximately 100uL of 36.6ng/uL plasmid sample A and approximately 100uL of 35.0ng/uL plasmid sample B.

A restriction digest will confirm that it is plasmid DNA not genomic DNA.

27th September 2010

Plasmid Prep using Qiagen Plasmid Midi Prep Kit (2nd attempt)

We attempted to purify the pET-3A vector again using the Qiagen Plasmid Midi Prep Kit as per the manufacturer’s protocols

The plasmid (two samples A & B) as well as some column flow-through collected at different stages of the purification were run on a GelRed post-stained 1.5% agarose gel and photo taken for visualization (see below).

Figure 11. Qiagen Plasmid Midi Prep results (2nd attempt)

Figure 11. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid purification. In lanes 1 and 10 there is a 1kb Molecular ladder. In lanes 2-9 there is the pooled plasmid purification samples that have been collected in separate tubes. All samples show a band indicative of linearized plasmid except lane 7 – this sample has been mistreated in the purification process.

Figure 12. Qiagen Plasmid Midi Prep results (2nd attempt)

Figure 12. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid flow through collections. In lanes 1 and 9 there is a 1kb Molecular ladder. In lanes 2 – 9 there are the column flow throughs that were collected at different points throughout the protocol. In lanes 2 and 3 there is a feint band visible but this is expected as it is sample collected before the addition of sample to the column.

Less DNA has been lost in the column flow through with this attempt however as we suspect that the plasmid is still contaminated with genomic DNA we decided to be safe and use another type of plasmid prep kit as well.

31st September 2010

Plasmid Prep using Invitrogen Midi Prep Kit

The pET-3A plasmid was purified using the Invitrogen Midi Prep Kit as per the manufacturer’s protocols

The plasmid (two samples 1 & 2) were run on a GelRed post-stained 1.5% agarose gel and photo taken for visualization (see below).

A band was seen for sample 2 but no band was observed for sample 2

This protocol has been successful but not enough plasmid has been purified

Figure 13. Plasmid Prep using Invitrogen Midi Prep Kit results

Figure 13. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid purification using Invitrogen Midi Prep kit. This kit shows that we have recovered less plasmid but it is more highly purified than what was purified with the other kits because the bands are less smeared.

31st September 2010

Restriction enzyme digest on Purified pET-3A

The enzyme digest was performed on the pET-3A vector that had been purified with the Qiagen Midi Prep protocol (performed on 27th September) as well as the pET-3A vector that had been purified with the Invitrogen Midi Prep kit (performed on the 31st September)

The pET-3A vector was digested with BamH1 and Nde1 restriction enzymes (see recognition sites below) so that the D. radiodurans and A. tumefaciens bacteriophytochrome-RBS-HO site operons that we have constructed can be ligated into the vector

The digests were run on a GelRed stained 1% agarose gel and photo taken for visualization (see below).

The restriction digest confirmed that it is plasmid DNA that has been purified is linearized and not circular as it collapsed to a single band of expected size and intensity

Restriction enzyme recognition sites:

Restriction enzyme	Recognition site
BamH1	5’-CA (cut) TATG – 3’ and 3’-CCTAG (cut) G-5’
Nde1	5’-CA(cut)TATG-3’and 3’-GTAT(cut)AG-5’

The reaction mastermix for the restriction digest to be performed on the Qiagen Midi Prep purified plasmid was set up as per the following recipe (per sample):

Mastermix:	Amount per sample (uL)
Gibco H2O	7.5
10x Buffer	10.0
BSA (10ug/uL)	0.5
pET-3A DNA	1.0
Restriction enzyme	1.0
Total	20.0

The reaction mastermix for the restriction digest to be performed on the Invitrogen Midi Prep kit purified plasmid was set up as per the following recipe (per sample):

Mastermix:	Amount per sample (uL)
Gibco H2O	2.5
10x Buffer	6.0
BSA (10ug/uL)	0.5
pET-3A DNA	50.0
Restriction enzyme	1.0
Total	20.0

Please note that 50uL of plasmid DNA was added to this reaction because we only obtained a low yield from the Invitrogen purification kit.

Figure 14. Restriction enzyme digest on Qiagen kit results:

Figure 14. GelRed post-stained 1% agarose gel of restriction enzyme digested with BamH1 and Nde1 of pET-3A plasmid purified with the Qiagen kits. Digest has been successful and shows that the purified plasmids have been linearized because they show a band of expected size and intensity.

Figure 15. Restriction enzyme digest on Invitrogen kit results:

Figure 15. GelRed post-stained 1% agarose gel of restriction enzyme digested with BamH1 and Nde1 of pET-3A plasmid purified with the Invitrogen kit. The samples have been pooled with this kit because there wasn’t much plasmid recovered. The enzyme digest was successful.

3rd October 2010

Plasmid Prep using Promega Wizard® Plus SV Minipreprs DNA Purification System

The pET-3A plasmid was purified using the Promega Wizard® Plus SV Minipreps DNA Purification System as per the manufacturer’s protocols

The only difference made to the protocol was that 50uL of Gibco H20 was added instead of 100uL for the elution step. This was labeled ‘F1’

Then 95uL Gibco H2O was added and collected. This was labeled ‘F2’

To try and obtain a higher yield the cell lysate was divided into 10 smaller tubes and a small column was used

The purification was run on a GelRed stained 1.5% agarose gel and photo taken for visualization (see below).

Figure 16. Promega Wizard® Plus SV Minipreps DNA Purification System results:

Figure 16. GelRed post-stained 1.5% agarose gel of pET-3A plasmid purification using Promega Wizard® Plus SV Minipreps DNA Purification System. In lanes 1 and 10 there is a 1kb Molecular ladder. In lanes 2-10 there is the 10 pooled cell lysate samples ‘F1’ that have been collected after 50uL of Gibco H20 was added. There is a band visible in each row from the first set of eluted samples.

Figure 17.

Figure 17. GelRed post-stained 1.5% agarose gel of pET-3A plasmid purification using Promega Wizard® Plus SV Minipreps DNA Purification System. In lanes 1 and 10 there is a 1kb Molecular ladder. In lanes 2-10 there is the 10 pooled cell lysate samples ‘F2’ that have been collected after additional 95uL of Gibco H2O was added. There is a feint band visible in each row from the second set of eluted samples.

3rd October 2010

Restriction enzyme digest of purified pET-3A vector

The enzyme digest was again performed on the pET-3A vector that had been purified with the Invitrogen Midi Prep kit (performed on the 31st September)

The enzyme digest was also performed on the pET-3A vector that had been purified with the Wizard® Plus SV Minipreps (performed on 3rd September)

The pET-3A vector was digested with BamH1 and Nde1 restriction enzymes so that the D. radiodurans and A. tumefaciens bacteriophytochrome-RBS-HO site operons that we have constructed can be ligated into the pET-3A vector

The digests were run on a GelRed stained 1.5% agarose gel and photo taken for visualization (see below).

The reaction mastermix for the restriction digest to be performed on the Invitrogen Midi Prep kit purified plasmid was set up as per the following recipe (per sample): Mastermix: Amount per sample (uL) Gibco H2O 2.5 10x Buffer 6.0 BSA (10ug/uL) 0.5 pET-3A DNA 50.0 Restriction enzyme 1.0 Total 20.0

Please note that 50uL of plasmid DNA was added to this reaction because we only obtained a low yield from the Invitrogen purification kit.

Figure 18.

Figure 18. GelRed post-stained 1.5% agarose gel of BamH1 and Nde1 restriction enzyme digested pET-3A purified using Wizard® Plus SV kit. In lane 1 and 13 there is a 1kb ladder. In lanes 2-6 there is the BamH1 digest. In lane 6 there is a gap. In lanes 7-11 there is the Nde1 digest. Digest has been successful and shows that it is plasmid DNA not genomic DNA that has been purified.

Figure 19

Figure 19. GelRed post-stained 1.5% agarose gel of BamH1 and Nde1 restriction enzyme digested pET-3A purified using Invitrogen kit. In lane 5 and 9 there is a 1kb ladder. In lane 6 there is the BamH1 digest. In lane 7 there is a gap. In lanes 8 there is the Nde1 digest. Digest has been successful and shows that it is plasmid DNA not genomic DNA that has been purified.

6th October 2010

Restriction enzyme digest of purified pET-3A vector obtained from various purification kits

As we needed approximately 200ng of plasmid for cloning the plasmid that had been with purified with various kits was pooled

The pooled purified plasmids were then digested with BamH1 and Nde1 restriction enzymes

The digests were run on a GelRed stained 1.5% agarose gel and photo taken for visualization (see below)

The reaction mastermix for the restriction digest to be performed on the Invitrogen Midi Prep kit purified plasmid was set up as per the following recipe (per sample):

Mastermix: Amount per sample (uL) Gibco H2O 3.0 10x Buffer 4.9 BSA (10ug/uL) 1.0 pET-3A DNA 30.0 Restriction enzyme 2.0 Total 20.0

Figure 20

Figure 20. GelRed post-stained 1.5% agarose gel of BamH1 and Nde1 restriction enzyme digested pET-3A purified using pooled samples purified with various kits. In lane 1 and 4 and 7 there is a 1kb ladder. In lane 2 there is the pooled Wizard product digested with BamH1. In lane 3 there is the pooled Qiagen product digested with BamH1. In lane 5 there is the pooled Wizard product digested with Nde1. In lane 6 there is the pooled Qiagen product digested with Nde1. Digest has been successful and shows that it is plasmid DNA not genomic DNA that has been purified.

6th October 2010

Ligation and plasmid clean up prior to cloning

The gene products were ligated into the pET-3A vector using the Promega T4 ligation system

After the restriction enzyme digest the products had to be cleaned of all excess salts and buffers

15th October 2010

Transformation into BL21(DE3) and Top 10 cells for pGEM-T Easy

The final products were transformed into two strains of E. coli

The first strain BL21(DE3) were used so that the HO gene could be expressed, if the transformation is successful green E. coli colonies will be observed

The top 10 cells were also transformed using blue/white screening methods as the pGEM-T Easy storage vector contains the lacZ gene.

Unfortunately none of the BL21(DE3) expressed the HO gene as all colonies grown were not green

The blue/white screening of the pGEM-T Easy clones were successful as there were a few clones that were white in colour (showing that the transformation had worked in these clones)

23rd/24th October 2010

Unfortunately, assembly of our biobrick part to submit to the registry was not successful when we followed the instructions given on the iGEM website (OR, our cells have mutated!!).

We have discovered that the linearised vector simply won’t cut with SpeI and we can only cut with EcoRI and PstI.

We have therefore suggested a new strategy – please see our ‘Safety’ page which shows the modification of the current protocol describing ligation in of a single Biobrick part with these two enzymes.

25th October 2010

Last attempt to assemble biobrick!

We have digested one of the Kan or Amp plasmids in the distribution kit that has a small part with XbaI and PstI and mix with EcoRI - SpeI digested pGEM-T-AT-Bph clones. We have then ligated with some of the linearised plasmid they supplied that we digest with PstI-EcoRI.

The following three restriction digests were set-up

EcoRI-PstI of the linearised vector.
SpeI 1hr followed by EcoRI 1hr of the AT-Bph gGEM-T clones 1 and 7. We did 5uL of each in 10uL digest volume.
XbaI-PstI digest of a part in well 23L in the distribution plate. Dissolve well contents in 10 uL water and digest 5 uL of this with XbaI and PstI. Total volume of digest was 10 ul. (It is not in the C3 vector. This part is just a double terminator part.)

Heat inactivate all then mix 3 uL of each, 1.1uL ligase buffer and 0.5 uL ligase. Ligate over lunch!! (2hr) and transform in the afternoon onto LB-Chloramphenicol plates.

26th October 2010
Biobrick assembly, phew!

We have about 12 transformants on the rbs-ATBph-terminator plates and on the AT-Bph-terminator plates!!

If these get inoculated this morning into LB-Chloramphenicol they should have grown enough by 4pm this afternoon to do minipreps.

We have inoculated 2 ml LB-Chloramphenicol with 10 of the #7 transformants and 1 of the #1 transformants. There may be a few others growing up but only these colonies were large enough to inoculate to liquid. Cells were put in shaking incubator for 7 hours for minipreping in the afternoon.

Restriction enzyme digest confirmed we have a biobrick!

Testing the ‘switch’ with biliverdin added to liquid cultures

Nothing like working to a deadline!!

The bacteriophytochrome containing cells were grown overnight in 5mL liquid culture (LB + amp) containing Biliverdin of varying concentrations and also IPTG to turn on expression of bacteriophytochrome, as follows:

The cells were tested for their ability to convert light using UV-Vis absorption spectroscopy as follows: YES IT WORKS = success!

100μL of overnight bacterial cultures were also run on a SDS PAGE. 100μL of the cells were centrifuged and the pellet resuspended in 40 μL SDS Page Sample Buffer (5 X concentrated). The cells were boiled for 2 minutes and loaded onto a 10% Bis-Tris Mini Gel (Invitrogen). Two gels were run, one with 8μL of the cells (low concentration) and one with 15μL of cells (high concentration). The SDS-PAGE gels were run at 180V for 35 minutes and stained in 10mM zinc acetate solution for approx 15 minutes.

Staining with zinc will induce fluorescence of the bound biliverdin chromophore to the bacteriophytochrome. The complex can be then visualized following SDS-PAGE electrophoresis using fluorescence. (See Figure 2 of Davis et al Science 286:2517 1999 http://www.scienceonline.org/cgi/reprint/286/5449/2517.pdf).

Figure 21: The gels were imaged for orange-red fluorescence using a Odyssey imager at 700nm.

Figure 21 Lane 1: empty; Lane 2: Benchmark Protein Ladder; Lanes 3-10: Bacterial samples #1, 2, 5, 6, 7, 8, 11, 12. Bacteriophytochrome – biliverdin complex in white box. Note, nice fluorescence for lanes 3 and 4 (high conc of BV), lower level of fluorescence of lane 5 (low conc of BV) and NO fluorescence of lane 6 (NO BV added). Same for lanes 7 to 9.

Figure 22

Figure 22: Following fluorescence visualization, the gels were stained with coomassie and the protein band confirmed to be ~80kDa which is the expected size of bacteriophytochrome.

Team:Macquarie Australia/Notebook3