Team:Newcastle University/Notebook

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{{:Team:Newcastle University/Header}}
{{:Team:Newcastle University/Header}}
{{:Team:Newcastle University/Template:UnderTheHome|page-title=[[Team:Newcastle University/Notebook|Wet Lab]]}}
{{:Team:Newcastle University/Template:UnderTheHome|page-title=[[Team:Newcastle University/Notebook|Wet Lab]]}}
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|title=Other progress
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|detail-text=[[Team:Newcastle University/Meetings|Meetings]]<br>[[Team:Newcastle University/Protocols|Protocols]]<br>[[Team:Newcastle University/Results|Results]]
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==Lab Notebook==
 
-
We finally started in the labs on Monday the 4th August.  So we will start updating the wiki with what's been happening and the progress we are making.
+
==Introduction==
-
[[Image:BB4.jpg]]
+
The overall design was too complicated for the time and resources available so we devised a proof of concept Brick which represented an input node from our full design. This Brick demonstrates that it is possible to transfer two-component peptide sensing from one strain into another and have it function as it did in the first strain. We took the subtilin two component system from ''Bacillus subtilis'' ATCC6633 and integrated it into the chromosome of ''B. subtilis'' 168, the typical laboratory ''B. subtilis'' strain at the ''amyE'' locus.
-
Fig 1: Newcastle iGEM team's construct
+
The wet lab work involved:
 +
* Cloning the synthesized Brick from pUC57 in ''E. coli'' into our ''Bacillus'' integration vectors (with GFP and mCherry respectively downstream of the Brick)
 +
* Validation of the integration vector
 +
* Transformation and integration into the chromosome of ''B. subtilis'' 168 at the ''amyE'' locus
 +
* Validation of chromosomal integration
 +
* Characterisation of the integrated parts and their response to subtilin.
-
Fig 1 shows the construct which contains:
+
 
 +
----
 +
 
 +
 
 +
Our subtilin sensor, part [http://partsregistry.org/Part:BBa_K104001 BBa_k104001]
 +
 
 +
[[Image:biobrickk.gif|thumb|center|300px|Figure 1: Newcastle's iGEM team construct.]]
 +
 +
Figure 1 shows the construct which contains:
* ''spaRK'' promotor
* ''spaRK'' promotor
-
* ''rrnB'' - rRNA binding site
+
* ''spaR'' (subtilin peptide antibiotic Regulation) - the 220 amino acid product of this gene usually regulates the downstream production of subtilin antibiotic. It has an N-terminal domain that can be phosphorylated and a C-terminal domian that has DNA binding properties [http://aem.asm.org/cgi/reprint/59/1/296.pdf]
-
* ''spaR'' (subtilin peptide antibiotic Regulation) - the 220 amino acid product of this gene usually regulates the downstream production of subtilin antibiotic. It has an N-terminal domain that can be phosphorylated and a C-terminal domian that has DNA binding properties [http://http://aem.asm.org/cgi/reprint/59/1/296.pdf]
+
* ''spaK'' (subtilin peptide antibiotic Kinase) - this gene codes for a 325 amino acid histadine kinase peptide that phosphorylates the N-terminus of ''spaR'' [http://aem.asm.org/cgi/reprint/59/1/296.pdf]. This activates the DNA binding ability of the C-terminus of ''spaR'', which in turn initiates transcription of the downstream gene. In the case of our construct, this gene is ''gfp''.
-
* ''spaK'' (subtilin peptide antibiotic Kinase) - this gene codes for a 325 amino acid histadine kinase peptide that phosphorylates the N-terminus of ''spaR'' [http://http://aem.asm.org/cgi/reprint/59/1/296.pdf]. This activates the DNA binding ability of the C-terminus of ''spaR'', which in turn initiates transcription of the downstream gene. In the case of our construct, this gene is ''gfp''.
+
* ''rrnO'' transcriptional terminator
-
* ''gfp'' (green fluorescence protein) - the marker being used to show activation of the ''spaRK'' system and therefore diagnosis of gram-positive bacteria by ''B. Subtilis''
+
* ''spaS'' promotor - a strong promotor inducible by upstream activation of ''spaRK''. It can be placed in front any gene to regulate its activity.
* ''spaS'' promotor - a strong promotor inducible by upstream activation of ''spaRK''. It can be placed in front any gene to regulate its activity.
 +
 +
 +
----
 +
'''Aim 1: clone the ''spaRK'' system from pUC57 into pJWV021 and transform into DH5 alpha competent ''E. coli'''''
'''Aim 1: clone the ''spaRK'' system from pUC57 into pJWV021 and transform into DH5 alpha competent ''E. coli'''''
-
[[Image:cloning 1.jpg]]
+
[[Image:cloning 1.jpg|thumb|center|300px|Aim 1]]
 +
 
 +
 
 +
----
 +
 
 +
'''Aim 2: clone the ''spaRK'' system from pUC57 into pGFP-rrnB and transform into DH5 alpha competent ''E. coli'''''
 +
 
 +
 
 +
[[Image:cloning 2.jpg|thumb|center|300px|Aim 2]]
 +
* The 2.2kb fragment (ncl08) contains the ''spaRK'' biobrick system. When properly cloned, our biobrick will replace the rrnB promoter in front of gfp present in plasmid pGFP-rrnB. Thus, gfp will now be under control of the subtilin inducible spaS promoter. This means that when ''SpaR'' is activated by SpaK (by sensing subtilin) its positive regulatory effect on PspaS will activate ''gfp''.
-
'''Aim 2: clone the ''spaRK'' system from pUC57 into pGFP-rrnB and transform into TOP10 competent ''E. coli'''''
 
-
[[Image:cloning 2.jpg]]
+
----
-
The 2.2kb fragment (ncl08) contains the ''spaRK'' system and promotor-less ''gfp'' linked to this. This means that when ''spaR'' is activated, its positive regulatory effect on spaK will in turn activate ''gfp''.
 
-
'''Aim 1: clone the ''agr'' system from the plasmid vector into pGFP-rrnB and transform into DH5 alpha competent ''E. coli'''''
 
 +
'''Aim 3: clone the ''agr'' (Cambridge biobrick for sensing S. aureus) system from the biobrick plasmid using EcoRI and SpeI sites into the corresponding sites of pGFP-rrnB. This will replace the PrrnB-gfp on the bacillus vector with the agr biobrick. The ligation was transformed into DH5 alpha competent ''E. coli'''''
-
[[Image:cloning 3.jpg]]
 
-
BBa_I647107 contains a partial ''agr'' operon, which includes ''agrC'' and ''A'' but has ''agrB'' and ''D'' deleted. This allows coding for the receptor (''C''/''A'') but not for production of the quorum peptides themselves (''B''/''D''). Eventually this will be linked to the ''spaRK'' system in a ''B. subtilis'' vector.
+
[[Image:cloning 3.jpg|thumb|center|300px|Aim 3]]
-
===Monday 4th August===
+
* BBa_I746107 contains a partial ''agr'' operon, which includes ''agrC'' and ''A'' but has ''agrB'' and ''D'' deleted. This allows coding for the receptor (''C''/''A'') but not for production of the quorum peptides themselves (''B''/''D''). The inducible promoter drives expression of gfp.
-
All 5 of us (Megan, Mark, Nina, Ria and Jess) went into the lab today to decide on a plan of action for the weeks to come. 
 
-
* We used electrophoresis to check for the presence of plasmids (pGFPrrnB - our plasmid with the ''gfp'' gene, and pJWV021 - with the ''mCherry'' gene).  Our gel was inconclusive, so we decided to re-run it the following day to confirm our results. 
 
-
* In the afternoon we attempted to transform TOP10 competent ''E. coli'' cells with DNA from the registry (from source plate 1010 well 4A and 1018 7A). 
+
<html><a name="subcloning"></html>
 +
== Subcloning ==
-
* We followed the extraction method outlined in the green folder and for each DNA spot we plated two petri dishes: one with a larger volume of DNA (4μl) and one with a smaller volume (2μl) to make colony counting easier.   We plated the following and incubated at 37˚C overnight:
+
The pUC57 was restriction-digested and the fragments displayed on a gel to ensure that the correct products have been produced.
-
'''Plate 1:''' +ve control (isolated plasmid plus TOP10 cells)
+
[[Image:02091.jpg|500px]]
 +
Figure 2: Gel from [[Newcastle_University_Wetlab/2_September_2008|2 Sept]] showing our 3 unrestricted plasmids, along with the 4 restrictions
 +
* '''Lane 1:''' 1kb ladder
 +
* '''Lane 2:''' Unrestricted pGFP-rrnB sample 1 (our bacillus integration vector that carries gfp between 2 amyE flanking regions)
 +
* '''Lane 3:''' Unrestricted pGFP-rrnB sample 2
 +
* '''Lane 4:''' Unrestricted pJWV021 sample 1 (our bacillus integration vector that carries mCherry between 2 sacA flanking regions)
-
'''Plate 2:''' -ve control (TOP10 cells only, no plasmid)
+
* '''Lane 5:''' Unrestricted pJWV021 sample 2
 +
* '''Lane 6:''' Unrestricted pUC57-ncl08 sample 1 (our synthetic biobrick construct; GenScript)
 +
* '''Lane 7:''' Unrestricted pUC57-ncl08 sample 2
 +
* '''Lane 8:''' 1kb ladder
 +
* '''Lane 9:''' pGFP-rrnB restricted with EcoR1 and Nhe1
 +
* '''Lane 10:''' pJWV021 restricted with BglII and Nhe1
 +
* '''Lane 11:''' pUC57-ncl08 restricted with EcoR1 and Nhe1
 +
* '''Lane 12:''' pUC57-ncl08 restricted with BglII and Nhe1
-
'''Plate 3:''' Large 1010 4A
+
Not shown here is the agr biobrick digestion.
-
'''Plate 4:''' Small 1010 4A
+
<html><a name="vector-validation"></html>
 +
== Validation of the integration vector ==
-
'''Plate 5:''' Large 1018 7A
 
-
'''Plate 6:''' Small 1018 7A
+
Digested pGFP-rrnB/pJWV021 were mixed with digested pUC57-ncl08 or agr biobrick, ligated and transformed to E.coli and the  bacteria [[Newcastle_University_Wetlab/4_September_2008|overnight]] were grown on selective media (the pGFP ligation on plates with spectinomycin (50microG/ml) and the pJWV021 ligation on kanamycin (10 microG/ml)). Transformants were grown up in LB with antibiotics, plasmid was isolated and digested to check for successful subcloning (in the GFP and mCherry vectors, GFP results shown below).
-
===Tuesday 5th August===
+
[[Image:Gel_2.jpg|500px]]
 +
Figure 4: Gel showing restricted pGFPrrnB-ncl08 taken from 12 different colonies
 +
* '''Lane 1:''' 1kb ladder
 +
* '''Lane 2:''' pGFPrrnB-ncl08 colony 1 (white colony, 25μl plate)
 +
* '''Lane 3:''' pGFPrrnB-ncl08 colony 2 (white colony, 25μl plate)
 +
* '''Lane 4:''' pGFPrrnB-ncl08 colony 3 (white colony, 25μl plate)
 +
* '''Lane 5:''' pGFPrrnB-ncl08 colony 4 (white colony, 25μl plate)
 +
* '''Lane 6:''' pGFPrrnB-ncl08 colony 5 (white colony, 25μl plate)
 +
* '''Lane 7:''' pGFPrrnB-ncl08 colony 6 (white colony, 25μl plate)
 +
* '''Lane 8:''' pGFPrrnB-ncl08 colony 7 (white colony, 25μl plate)
 +
* '''Lane 9:''' pGFPrrnB-ncl08 colony 8 (white colony, 25μl plate)
 +
* '''Lane 10:''' pGFPrrnB-ncl08 colony 9 (white colony, 25μl plate)
 +
* '''Lane 11:''' pGFPrrnB-ncl08 colony 10 (white colony, 250μl plate)
 +
* '''Lane 12:''' pGFPrrnB-ncl08 colony 11 (white colony, 250μl plate)
 +
* '''Lane 13:''' pGFPrrnB-ncl08 colony 12 (green colony, 25μl plate)
-
Today we re-ran the gel electrophoresis of pGFP-rrnB and pJWV021 to check correct plasmid size and sufficient plasmid quantity. Results showed this to be true and the expected plasmid sizes of 8399bp for pGFP-rrnB and 7066bp for pJWV021.
+
This demonstrated that we had succesfully subcloned our ncl08 biobrick from pUC57-ncl08 to pGFP. Not shown here are the restriction checks for pJWV021-ncl08 and pGFP-agr (but we have correct clones for them as well!).  
-
===Wednesday 6th August===
 
-
Analyses of results from Monday’s transformation showed no colony growth except for the positive control. We decided to try another transformation into TOP10 E. coli using different spots from the registry. This time we used 1018 7A and 1004 6G.  
+
<html><a name="bsubtilis-integration"></html>
 +
==Transformation and integration into the ''B. subtilis'' chromosome==
-
We noticed that Cambridge, who had also been having problems, had adapted the extraction method.  So we tried their improved method, with a couple of minor differences:
+
[[Image:CloningStratStep2.jpg|500px|thumb|left]]
-
'''1)'''  Warm 50μl of H20 in an Eppendorf tube at 50˚C
 
-
'''2)'''  Add 4 punched-out spots from the registry
+
<html><a name="chromosome-validation"></html>
 +
==Validation of chromosomal integration==
-
'''3)'''  Keep at 50˚C for 20 minutes
+
Next, we transformed the correct plasmids to (self made) competent ''Bacillus'', as described in the protocol section. For plasmid pGFP-ncl08 and pGFP-agr, bacillus was plated on chloramphenicl (5 microG/ml) and for plasmid pJWV021-ncl08, bacillus were plated on kanamycin (5 microG/ml). Next day, Bacillus transformants were subseqently streaked to single colonies on selective agar plates. To check for correct integration of the pGFP-ncl08 and pGFP-agr plasmids at the amyE locus, single colonies were streaked on nutrient agar plates containing 1% starch. If a double crossover event occured, cells are not able to produce AmyE and will not degrade starch, thus no 'halo' around the colony. 2 individual colonies without an halo were grown and stocked and used for later analyses. To check correct integration of the pJWV021-ncl08 plasmid in the Bacillus chromosome, single colonies were streaked on minimal medium plates containing either glucose or sucrose as the sole carbon source. If the plasmid integrated correctly at the sacA locus via a double crossover, cells cannot utilise sucrose anymore and will not grow on the minimal plate containing sucrose, but will grow on the glucose plate. Again, 2 correct transformants were selected that were unable to proliferate on sucrose plates and stocked in the -80C freezer for future work.
-
'''4)'''  Centrifuge at 13,300g for 3 minutes
 
-
'''5)'''  Warm for a further 10 minutes at 50˚C
 
-
'''6)'''  Centrifuge at 13,300g for 3 minutes
+
<html><a name="characterisation"></html>
 +
==Characterisation of the integrated parts and their response to subtilin==
-
'''7)'''  Pipette out the supernatant which should (hopefully!) contain the DNA
+
<html><a name="results"></html>
 +
== Results ==
 +
To analyse the results of the wet lab transformations of the inserts into ''B. subtilis'', we used two methods: microscopy and flow cytometry.
-
To see the original modified method, go to Cambridge’s OpenWetWare page:
+
===Microscopy===
-
http://openwetware.org/wiki/IGEM:Cambridge/2008/Protocols
+
-
===Thursday 7th August===
+
Microscopy work from 08.09.08 showed a difference in the level of flourescence of the iGEMgfp fluorescent cells (higher in 10% subtilin-induced cells compared to 0% subtilin-induced cells). However, there was little difference in the ''number'' of cells that fluoresced between the two cultures.
-
Unfortunately none of the plates showed any colonies. However, further incubation overnight of the plates at 37˚C from Monday 4th August produced 5 colonies on Plate 4 (2μl 1010 4A). These colonies were individually incubated overnight at 37˚C in 10μl LB.
+
There was no difference in the number of fluorecent cells ''or'' the level of fluorescence between the 10% subtilin-induced and the 0% subtilin-induced iGEMcherry cells.
-
===Friday 8th August===
 
-
The 5 colonies from Thursday 7th August cultured in LB were pooled and the plasmid purified. When the sample was run on gel no band appeared, showing that the colonies were not ''E. coli'' and must have resulted from contamination.
+
===Flow cytometry===
-
===Monday 11th August===
+
Flow cytometry measures cell fluorescence and light scattering, cell-by-cell, allowing us to quantify our results and present them in graphical form.  A sample of our cells engineered ''B. subtilis'' cells were injected into the machine, which hydro-dynamically focusses the fluid. Lasers of a particular wavelength are directed onto the stream of fluid, and each particle which passes through the light beam will cause the laser to scatter in a different way. Cells which absorb and then re-emit the light, result in fluorescence (a higher energy state).
-
Plasmids were isolated from pUc57-ncl08 (the ncl08 fragment contains the ''spaRK'' 2-part component system). We aim to clone this into pGFP-rrnB and pJWV021 vectors.
+
The detectors in the machine measure the scattering of light and any fluoresence which occurs, and the data is displayed as histograms and dot plots.
-
Restrictions of pGFP-rrnB and pJWV021 carried out using 10μl plasmid in a total volume of 50μl. pJWV021 was restricted in a 2-step reaction as we beleived that the enzymes were incompatible in the same buffer. The second restriction of this plasmid used 48μl of the purified plasmid mixture in a total volume of 100μl. pGFP-rrnB was restricted using EcoRI and NHeI. pJWV021 was restricted firstly with NHeI and secondly with BglII.
+
'''Induction by 0% v/v subtilin''' (i.e in the absence of subtilin): mean fluoresence = 7.70
-
These were purified and stored overnight at -25˚C.
+
'''Induction by 1% v/v subtilin:''' mean fluoresence = 14.77
-
===Wednesday 27th August===
+
'''Induction by 10% v/v subtilin:''' the mean fluoresence = 21.95
-
Overnight cultures were analysed:
+
These results show that the higher the concentration of subtilin, the more GFP is expressed.
-
- pGFP-rrnB grown on ampicillin - cells had lysed (medium was clear with large bubbles at the surface).
+
-
- pJWV021 grown on ampicillin - frozen glycerol stock made (see protocols section).
+
-
- pUC57-ncl08 grown on ampicillin - frozen glycerol stock made.
+
-
A colony stab was taken of pGFP-rrnB using 10μl spectinomycin in 10mL LB as the culture medium.   
+
At 10% induction, the subtilin began to cause some of the cells to lyse - this can be seen by the two distinct populations of cells - one being the cells which have lysed, and the other being the remainder of intact cells.  Since subtilin is an antibiotic, this was a good indication that the subtilin we had collected that day was of good quality.
-
===Thursday 28th August===
+
Overall, the microscopy and flow cytometry suggest that our biobrick is functional, but requires high levels of subtilin in order to be activated. The days that we performed microscopy for instance, we did not observe killing of the cells after 10% addition of subtilin, indicating that our subtilin for that day was at a low concentration. Subsequently, we did not observe a strong GFP induction. However, when we did observe killing (indicative of high subtilin concentrations), we did observe induction of GFP (see flow cytometry). So in principle the system works, and we have engineered the lab strain of ''B. subtilis'' 168 to respond to the external addition of subtilin, but more work should be done to further optimise the system. For instance, it was shown that the spaS promoter fused to gfp gives high yield of protein production when present on a multicopy plasmid, after the addition of subtilin (Bongers et al, AEM). Also it may be possible to introduce a positive feedback effect by introducing a ''spaS'' box and associated promoter upstream of ''spaRK'' in addition to the normal promoter.
-
New frozen glycerol stock of cells made to be kept at -80˚C.
+
<gallery>
 +
Image:Montage.jpg|Microscopy results of gfp upon 1% induction by subtilin
 +
Image:Montage_pc.jpg|Microscopy results of mCherry upon 1% induction by subtilin
 +
Image:Flow0%.jpg|Flow cytometry results for 0% induction by subtilin.
 +
Image:Flow1%.jpg|Flow cytometry results for 1% induction by subtilin.
 +
Image:Flow10%.jpg|Flow cytometry results for 10% induction by subtilin.
 +
</gallery>
-
Isolated plasmid from overnight culture of pGFP-rrnB. For this the 10mL culture was divided into 5 x 2mL plastic tubes and pelleted by centrifugation for 10 minutes. The supernatent was discarded and follwing the isolation procedure the product was pooled.
 
-
===Friday 29th August===
 
-
Today we digested our pUC57-ncl08 plasmid.  In one reaction we used EcoR1 and Nhe1, and in other we used BglII and Nhe1.  We also carried out a digestion with just BglII to check for the correct sized linear DNA.  In addition we ran undigested plasmid on the gel.
+
<html><a name="protocols"></html>
 +
===Protocols===
-
              '''Expected band sizes:'''
+
Listed below are the lab protocols that were developed or used for the Newcastle University iGEM 2008 wet lab component. Please click on any item for detailed instructions.
-
              * For digestion with BglII only: 4.9kb
+
-
              * For digestion with Nhe1 and EcoR1: 2.2kb and 2.7kb
+
-
              * For digestion with Nhe1 and BglII: 2.2kb and 2.7kb
+
-
As you can see, the digests appear successful. 
+
* [[Agarose Gel Electrophoresis]]
-
[[Image:gelFINAL.jpg]]
+
* [[Making Agar Plates]]
 +
* [[Isolating Plasmid from Cells (Miniprep)]]
 +
* [[Restricting Plasmids (Double Restriction)]]
 +
* [[Purifying DNA from an enzymatic reaction]]
 +
* [[Cutting a Specific Band from Agarose Gel]]
 +
* [[Purifying DNA from Gel Slices]]
 +
* [[Ligating DNA]]
 +
* [[Transforming into DH5α or TOP10 E. coli|Transforming into DH5α or TOP10 ''E. coli'']]
 +
* [[Making Frozen Glycerol Cell Stocks From Overnight Cultures]]
 +
* [[Making Overnight Cultures from Frozen Glycerol Cell Stocks]]
 +
* [[Making Overnight Cultures from Agar Colonies]]
 +
* [[Transforming into Bacillus subtillis|Transforming into ''Bacillus subtilis'']]
 +
* [[Inducing Bacillus subtilis with Subtilin|Inducing ''Bacillus subtilis'' with Subtilin]]
 +
* [[Preparing Bacillus subtilis for Microscopy|Preparing ''Bacillus subtilis'' for Microscopy]]
-
===Thursday 04th September===
 
-
The plates from 03.09.08 showed expected colony growth and therefore stab cultures were taken from individual colonies on Plates 1, 2 and 9. The culture media were made using 10mL of LB in 15mL plastic tubes together with the correct volume of the relavent antibiotic. This was 5μl spectinomycin for the GFP-rrnB colonies and 4μl kanomycin for the pJWV021 colonies. Cultures were taken as follows:
 
-
'''GFP-rrnB - pUC57 ligation (grown on spec)'''
+
<html><a name="journal"></html>
-
* 9 white colonies (Plate 1)
+
== Wet Lab Journal ==
-
* 1 green colony (Plate 1)
+
-
* 2 white colonies (Plate 2)
+
-
'''pJWV021 - pUC57 ligation (grown on kan)'''
+
This contains the day-to-day progress of our wet lab team. Click on a day below to find more details of the work above.
-
* 10 white colonies (Plate 9)
+
-
* 1 pink colony (Plate 9)
+
-
* 1 bright white colony (Plate 9) (this is likely to a be contaminant non-E.coli colony)
+
-
All 24 culture tubes were incubated at 37˚C whilst shaking for ~20 hours. This large quantity of cultures should ensure that we obtain at least 2 cultures (one GFP-rrnB and one pJWV021) that have taken up the ncl08 insert. The plates were kept at room temperature in case fulrther stab cultures needed to be made.
+
{|cellpadding="10"
 +
|{{#calendar: title=Newcastle University Wetlab |year=2008 | month=08 }}
 +
|{{#calendar: title=Newcastle University Wetlab |year=2008 | month=09 }}
 +
|}
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</div><!--maincontent -->
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</div><!--main col -->

Latest revision as of 01:53, 30 October 2008

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Newcastle University

GOLD MEDAL WINNER 2008

Home Team Original Aims Software Modelling Proof of Concept Brick Wet Lab Conclusions


Home >> Wet Lab

Introduction

The overall design was too complicated for the time and resources available so we devised a proof of concept Brick which represented an input node from our full design. This Brick demonstrates that it is possible to transfer two-component peptide sensing from one strain into another and have it function as it did in the first strain. We took the subtilin two component system from Bacillus subtilis ATCC6633 and integrated it into the chromosome of B. subtilis 168, the typical laboratory B. subtilis strain at the amyE locus.

The wet lab work involved:

  • Cloning the synthesized Brick from pUC57 in E. coli into our Bacillus integration vectors (with GFP and mCherry respectively downstream of the Brick)
  • Validation of the integration vector
  • Transformation and integration into the chromosome of B. subtilis 168 at the amyE locus
  • Validation of chromosomal integration
  • Characterisation of the integrated parts and their response to subtilin.



Our subtilin sensor, part [http://partsregistry.org/Part:BBa_K104001 BBa_k104001]

Figure 1: Newcastle's iGEM team construct.

Figure 1 shows the construct which contains:

  • spaRK promotor
  • spaR (subtilin peptide antibiotic Regulation) - the 220 amino acid product of this gene usually regulates the downstream production of subtilin antibiotic. It has an N-terminal domain that can be phosphorylated and a C-terminal domian that has DNA binding properties [http://aem.asm.org/cgi/reprint/59/1/296.pdf]
  • spaK (subtilin peptide antibiotic Kinase) - this gene codes for a 325 amino acid histadine kinase peptide that phosphorylates the N-terminus of spaR [http://aem.asm.org/cgi/reprint/59/1/296.pdf]. This activates the DNA binding ability of the C-terminus of spaR, which in turn initiates transcription of the downstream gene. In the case of our construct, this gene is gfp.
  • rrnO transcriptional terminator
  • spaS promotor - a strong promotor inducible by upstream activation of spaRK. It can be placed in front any gene to regulate its activity.



Aim 1: clone the spaRK system from pUC57 into pJWV021 and transform into DH5 alpha competent E. coli


Aim 1


Aim 2: clone the spaRK system from pUC57 into pGFP-rrnB and transform into DH5 alpha competent E. coli


Aim 2
  • The 2.2kb fragment (ncl08) contains the spaRK biobrick system. When properly cloned, our biobrick will replace the rrnB promoter in front of gfp present in plasmid pGFP-rrnB. Thus, gfp will now be under control of the subtilin inducible spaS promoter. This means that when SpaR is activated by SpaK (by sensing subtilin) its positive regulatory effect on PspaS will activate gfp.



Aim 3: clone the agr (Cambridge biobrick for sensing S. aureus) system from the biobrick plasmid using EcoRI and SpeI sites into the corresponding sites of pGFP-rrnB. This will replace the PrrnB-gfp on the bacillus vector with the agr biobrick. The ligation was transformed into DH5 alpha competent E. coli


Aim 3
  • BBa_I746107 contains a partial agr operon, which includes agrC and A but has agrB and D deleted. This allows coding for the receptor (C/A) but not for production of the quorum peptides themselves (B/D). The inducible promoter drives expression of gfp.


Subcloning

The pUC57 was restriction-digested and the fragments displayed on a gel to ensure that the correct products have been produced.

02091.jpg Figure 2: Gel from 2 Sept showing our 3 unrestricted plasmids, along with the 4 restrictions

  • Lane 1: 1kb ladder
  • Lane 2: Unrestricted pGFP-rrnB sample 1 (our bacillus integration vector that carries gfp between 2 amyE flanking regions)
  • Lane 3: Unrestricted pGFP-rrnB sample 2
  • Lane 4: Unrestricted pJWV021 sample 1 (our bacillus integration vector that carries mCherry between 2 sacA flanking regions)
  • Lane 5: Unrestricted pJWV021 sample 2
  • Lane 6: Unrestricted pUC57-ncl08 sample 1 (our synthetic biobrick construct; GenScript)
  • Lane 7: Unrestricted pUC57-ncl08 sample 2
  • Lane 8: 1kb ladder
  • Lane 9: pGFP-rrnB restricted with EcoR1 and Nhe1
  • Lane 10: pJWV021 restricted with BglII and Nhe1
  • Lane 11: pUC57-ncl08 restricted with EcoR1 and Nhe1
  • Lane 12: pUC57-ncl08 restricted with BglII and Nhe1

Not shown here is the agr biobrick digestion.

Validation of the integration vector

Digested pGFP-rrnB/pJWV021 were mixed with digested pUC57-ncl08 or agr biobrick, ligated and transformed to E.coli and the bacteria overnight were grown on selective media (the pGFP ligation on plates with spectinomycin (50microG/ml) and the pJWV021 ligation on kanamycin (10 microG/ml)). Transformants were grown up in LB with antibiotics, plasmid was isolated and digested to check for successful subcloning (in the GFP and mCherry vectors, GFP results shown below).

Gel 2.jpg Figure 4: Gel showing restricted pGFPrrnB-ncl08 taken from 12 different colonies

  • Lane 1: 1kb ladder
  • Lane 2: pGFPrrnB-ncl08 colony 1 (white colony, 25μl plate)
  • Lane 3: pGFPrrnB-ncl08 colony 2 (white colony, 25μl plate)
  • Lane 4: pGFPrrnB-ncl08 colony 3 (white colony, 25μl plate)
  • Lane 5: pGFPrrnB-ncl08 colony 4 (white colony, 25μl plate)
  • Lane 6: pGFPrrnB-ncl08 colony 5 (white colony, 25μl plate)
  • Lane 7: pGFPrrnB-ncl08 colony 6 (white colony, 25μl plate)
  • Lane 8: pGFPrrnB-ncl08 colony 7 (white colony, 25μl plate)
  • Lane 9: pGFPrrnB-ncl08 colony 8 (white colony, 25μl plate)
  • Lane 10: pGFPrrnB-ncl08 colony 9 (white colony, 25μl plate)
  • Lane 11: pGFPrrnB-ncl08 colony 10 (white colony, 250μl plate)
  • Lane 12: pGFPrrnB-ncl08 colony 11 (white colony, 250μl plate)
  • Lane 13: pGFPrrnB-ncl08 colony 12 (green colony, 25μl plate)

This demonstrated that we had succesfully subcloned our ncl08 biobrick from pUC57-ncl08 to pGFP. Not shown here are the restriction checks for pJWV021-ncl08 and pGFP-agr (but we have correct clones for them as well!).


Transformation and integration into the B. subtilis chromosome

CloningStratStep2.jpg


Validation of chromosomal integration

Next, we transformed the correct plasmids to (self made) competent Bacillus, as described in the protocol section. For plasmid pGFP-ncl08 and pGFP-agr, bacillus was plated on chloramphenicl (5 microG/ml) and for plasmid pJWV021-ncl08, bacillus were plated on kanamycin (5 microG/ml). Next day, Bacillus transformants were subseqently streaked to single colonies on selective agar plates. To check for correct integration of the pGFP-ncl08 and pGFP-agr plasmids at the amyE locus, single colonies were streaked on nutrient agar plates containing 1% starch. If a double crossover event occured, cells are not able to produce AmyE and will not degrade starch, thus no 'halo' around the colony. 2 individual colonies without an halo were grown and stocked and used for later analyses. To check correct integration of the pJWV021-ncl08 plasmid in the Bacillus chromosome, single colonies were streaked on minimal medium plates containing either glucose or sucrose as the sole carbon source. If the plasmid integrated correctly at the sacA locus via a double crossover, cells cannot utilise sucrose anymore and will not grow on the minimal plate containing sucrose, but will grow on the glucose plate. Again, 2 correct transformants were selected that were unable to proliferate on sucrose plates and stocked in the -80C freezer for future work.


Characterisation of the integrated parts and their response to subtilin

Results

To analyse the results of the wet lab transformations of the inserts into B. subtilis, we used two methods: microscopy and flow cytometry.

Microscopy

Microscopy work from 08.09.08 showed a difference in the level of flourescence of the iGEMgfp fluorescent cells (higher in 10% subtilin-induced cells compared to 0% subtilin-induced cells). However, there was little difference in the number of cells that fluoresced between the two cultures.

There was no difference in the number of fluorecent cells or the level of fluorescence between the 10% subtilin-induced and the 0% subtilin-induced iGEMcherry cells.


Flow cytometry

Flow cytometry measures cell fluorescence and light scattering, cell-by-cell, allowing us to quantify our results and present them in graphical form. A sample of our cells engineered B. subtilis cells were injected into the machine, which hydro-dynamically focusses the fluid. Lasers of a particular wavelength are directed onto the stream of fluid, and each particle which passes through the light beam will cause the laser to scatter in a different way. Cells which absorb and then re-emit the light, result in fluorescence (a higher energy state).

The detectors in the machine measure the scattering of light and any fluoresence which occurs, and the data is displayed as histograms and dot plots.

Induction by 0% v/v subtilin (i.e in the absence of subtilin): mean fluoresence = 7.70

Induction by 1% v/v subtilin: mean fluoresence = 14.77

Induction by 10% v/v subtilin: the mean fluoresence = 21.95

These results show that the higher the concentration of subtilin, the more GFP is expressed.

At 10% induction, the subtilin began to cause some of the cells to lyse - this can be seen by the two distinct populations of cells - one being the cells which have lysed, and the other being the remainder of intact cells. Since subtilin is an antibiotic, this was a good indication that the subtilin we had collected that day was of good quality.

Overall, the microscopy and flow cytometry suggest that our biobrick is functional, but requires high levels of subtilin in order to be activated. The days that we performed microscopy for instance, we did not observe killing of the cells after 10% addition of subtilin, indicating that our subtilin for that day was at a low concentration. Subsequently, we did not observe a strong GFP induction. However, when we did observe killing (indicative of high subtilin concentrations), we did observe induction of GFP (see flow cytometry). So in principle the system works, and we have engineered the lab strain of B. subtilis 168 to respond to the external addition of subtilin, but more work should be done to further optimise the system. For instance, it was shown that the spaS promoter fused to gfp gives high yield of protein production when present on a multicopy plasmid, after the addition of subtilin (Bongers et al, AEM). Also it may be possible to introduce a positive feedback effect by introducing a spaS box and associated promoter upstream of spaRK in addition to the normal promoter.

Microscopy results of gfp upon 1% induction by subtilin

Microscopy results of mCherry upon 1% induction by subtilin

Flow cytometry results for 0% induction by subtilin.

Flow cytometry results for 1% induction by subtilin.

Flow cytometry results for 10% induction by subtilin.


Protocols

Listed below are the lab protocols that were developed or used for the Newcastle University iGEM 2008 wet lab component. Please click on any item for detailed instructions.


Wet Lab Journal

This contains the day-to-day progress of our wet lab team. Click on a day below to find more details of the work above.

August
MTWTFSS
        [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/1_August_2008&action=edit 1] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/2_August_2008&action=edit 2] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/3_August_2008&action=edit 3]
[http://2008.igem.org/Newcastle_University_Wetlab/4_August_2008 4] [http://2008.igem.org/Newcastle_University_Wetlab/5_August_2008 5] [http://2008.igem.org/Newcastle_University_Wetlab/6_August_2008 6] [http://2008.igem.org/Newcastle_University_Wetlab/7_August_2008 7] [http://2008.igem.org/Newcastle_University_Wetlab/8_August_2008 8] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/9_August_2008&action=edit 9] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/10_August_2008&action=edit 10]
[http://2008.igem.org/Newcastle_University_Wetlab/11_August_2008 11] [http://2008.igem.org/Newcastle_University_Wetlab/12_August_2008 12] [http://2008.igem.org/Newcastle_University_Wetlab/13_August_2008 13] [http://2008.igem.org/Newcastle_University_Wetlab/14_August_2008 14] [http://2008.igem.org/Newcastle_University_Wetlab/15_August_2008 15] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/16_August_2008&action=edit 16] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/17_August_2008&action=edit 17]
[http://2008.igem.org/Newcastle_University_Wetlab/18_August_2008 18] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/19_August_2008&action=edit 19] [http://2008.igem.org/Newcastle_University_Wetlab/20_August_2008 20] [http://2008.igem.org/Newcastle_University_Wetlab/21_August_2008 21] [http://2008.igem.org/Newcastle_University_Wetlab/22_August_2008 22] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/23_August_2008&action=edit 23] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/24_August_2008&action=edit 24]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/25_August_2008&action=edit 25] [http://2008.igem.org/Newcastle_University_Wetlab/26_August_2008 26] [http://2008.igem.org/Newcastle_University_Wetlab/27_August_2008 27] [http://2008.igem.org/Newcastle_University_Wetlab/28_August_2008 28] [http://2008.igem.org/Newcastle_University_Wetlab/29_August_2008 29] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/30_August_2008&action=edit 30] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/31_August_2008&action=edit 31]
September
MTWTFSS
[http://2008.igem.org/Newcastle_University_Wetlab/1_September_2008 1] [http://2008.igem.org/Newcastle_University_Wetlab/2_September_2008 2] [http://2008.igem.org/Newcastle_University_Wetlab/3_September_2008 3] [http://2008.igem.org/Newcastle_University_Wetlab/4_September_2008 4] [http://2008.igem.org/Newcastle_University_Wetlab/5_September_2008 5] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/6_September_2008&action=edit 6] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/7_September_2008&action=edit 7]
[http://2008.igem.org/Newcastle_University_Wetlab/8_September_2008 8] [http://2008.igem.org/Newcastle_University_Wetlab/9_September_2008 9] [http://2008.igem.org/Newcastle_University_Wetlab/10_September_2008 10] [http://2008.igem.org/Newcastle_University_Wetlab/11_September_2008 11] [http://2008.igem.org/Newcastle_University_Wetlab/12_September_2008 12] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/13_September_2008&action=edit 13] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/14_September_2008&action=edit 14]
[http://2008.igem.org/Newcastle_University_Wetlab/15_September_2008 15] [http://2008.igem.org/Newcastle_University_Wetlab/16_September_2008 16] [http://2008.igem.org/Newcastle_University_Wetlab/17_September_2008 17] [http://2008.igem.org/Newcastle_University_Wetlab/18_September_2008 18] [http://2008.igem.org/Newcastle_University_Wetlab/19_September_2008 19] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/20_September_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/21_September_2008&action=edit 21]
[http://2008.igem.org/Newcastle_University_Wetlab/22_September_2008 22] [http://2008.igem.org/Newcastle_University_Wetlab/23_September_2008 23] [http://2008.igem.org/Newcastle_University_Wetlab/24_September_2008 24] [http://2008.igem.org/Newcastle_University_Wetlab/25_September_2008 25] [http://2008.igem.org/Newcastle_University_Wetlab/26_September_2008 26] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/27_September_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/28_September_2008&action=edit 28]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/29_September_2008&action=edit 29] [http://2008.igem.org/Newcastle_University_Wetlab/30_September_2008 30]
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