Team:University College London/LabBook/Week8

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== Monday 30.6.12 ==

'''Aim:''' Repeat the gel from Expt 7.3 (week 7) with a smaller ladder and higher agarose percentage in order to detect the very small inserts of BBa_J23119 (35bp) and BBa_B0034 (12hp). Previous ladder used was 10037 bp, and this time we used a 25bp ladder.

<html><div class="protocol protocol-RestrictionEnzymeDigest">Restriction Enzyme Digest Protocol 1</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/RestrictionEnzymeDigest1}}<html></div></html>

'''Step 2 - Setting up Digests and Controls:''' The protocol describes the recipe for (i) Digested Plasmid and (ii) Uncut Control.  The table below indicates that an uncut and an Xba1/Spe1 digested sample be set up for each BioBrick. Set up Eppendorfs as follows


{| class="wikitable"
|-
! colspan="2" |  Samples  !!Recipe !! Enzymes 
|-
| rowspan="7" |BioBrick || rowspan="2" | BBa_J23119 ||Digested Plasmid || Xba1 & Spe1 
|-
| Undigested Plasmid (Control) || None 
|-
| rowspan="2" | BBa_B0034 || Digested Plasmid|| Xba1 & Spe1
|-
| Undigested Plasmid (Control) || None 
|-
|}


<html><div class="protocol protocol-Electrophoresis">Electrophoresis Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Electrophoresis}}<html></div></html>

'''Results:'''The image below shows a 3.5% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 7.3. The high agarose concentration of the gel was intended to slow the progression of DNA fragments to enable us to detect the small inserts of BBa_J23119 and BBa_B0034. The table below indicates the expected sizes of BioBricks and Plasmid Backbones. Accordingly, we used a the smallest available ladder, with fragments ranging from 25bp to 500bp. At the top of the gel it is possible to see the large fragments we detected on a 1000bp gel in Expt 7.3 Week 7. However, we had expected this gel to also demonstrate the BBa_J23119 insert (35bp) corresponding to letter A in Lane 1, and the BBa_B0034 insert, corresponding to letter B in Lane 3. No product was found. As expected, there was no product measurable against the 25bp ladder for the uncut plasmids in Lane 2 and Lane 4. 

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{| class="wikitable"
|-
! BioBrick !! Expected BioBrick Size (bp) !! Plasmid Backbone !! Plasmid Expected Size (bp) !! Combined Size
|-
| BBa_J23119 || 35 || PSB1A2 || 2079 || 2114
|-
| BBa_B0034 || 12|| PSB1A2 || 2079|| 2091
|}


'''Conclusion:'''No product was detected for either BioBrick insert. However, we do no feel this necessarily proves the transformation failed, especially as we detected the correct plasmid backbone for each in week 7 (Expt 7.3). The modest nanodrop concentration obtained last week suggests that the concentration was not high enough to allow such a small fragment to be visualised. We are considering alternatives such as running a polyacrylamide gel, which is more sensitive to small fragments, or amplifying the fragment using PCR

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== Monday 30.7.12 ==

'''Aim - Picking colonies:''' Colonies were picked for BBa_I750016, which demonstrated colony formation in Expt 7.4 on Friday 27.7.12. 
 
<html><div class="protocol protocol-ColPic">Picking Colonies</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/ColPic}}<html></div></html>

'''Step 2 – Inoculating Colonies into a Selective Broth:''' The table below indicates the volume of broth and the concentration of antibiotic required for each BioBrick.

{| class="wikitable"
|-
! colspan="2" |  Samples !! Volume Inoculated !! Broth (ml) !! Antibiotic (ug/ml)
|-
| rowspan="8" |BioBrick ||rowspan="2" | BBa_I750016  || 10ul ||rowspan="8" |Lysogeny Broth (5) || rowspan="8" | Ampicillin(50ug/ml)
|-
| 90ul 
|}


== Tuesday 31.7.12 ==


'''Aim – Results from Colony Picking'''

'''Results:''' The table below indicates whether there was growth for BBa_I750016


{| class="wikitable"
|-
! colspan="2" |  Samples !! Volume Inoculated !! Colony Formation
|-
| rowspan="8" |BioBrick ||rowspan="2" | BBa_I750016  || 10μl || Yes
|-
| 90μl || Yes
|}

'''Conclusion:''' We can proceed onto Miniprep, Analytical Digest, and Nanodrop.


'''Method'''

'''Miniprep:''' 
<html><div class="protocol protocol-Miniprep">Miniprep Protocol 1 (ANACHEM)</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Miniprep2}}<html></div></html>


== Wednesday 1.8.12 ==

Aim: Nanodrop of BBa_I750016

{| class="wikitable"
|-
! BioBrick !! λ260 !! λ 280
|-
| BBa_I750016 (ng/μl) || 7.6 || 7.6
|-
|}

'''Conclusion:''' The concentration of BioBrick is to low to justify running a gel. We conclude the transformation failed.

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== Monday 30.7.12 ==

'''Aim - Transformation of previously failed BioBricks:''' BBa_C0040 was transformed in Expt 7.4, but appeared to be contaminated. BBa_R0040, as part of the same experiment, did not form colonies at all after transformation. Therefore both were reattempted.


'''Method:'''
<html><div class="protocol protocol-Transformation">Transformation Protocol 1</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Transformation1}}<html></div></html>


'''Step 1 – Thawing Cells:''' Use W3110 cell line created in Week 2 (Expt 2.1)

'''Step 3 – Addition of BioBrick:''' To each 2ml eppendorf, add 1ul of the following BioBricks. Include an extra tube as a control, with no BioBrick added

{| class="wikitable"
|-
!  colspan="2" |   !! Function !! Module
|-
| rowspan="2" |BioBrick|| BBa_C0040 || Tetracycline Repressor|| Buoyancy
|-
| BBa_R0040 || TetR Repressible Promoter || Buoyancy
|-
| rowspan="2"|Control || Positive (one for each of the above BioBricks)|| ||
|-
| Negative (No BioBrick) || ||
|}

'''Step 9 – Plating samples on Agar Plates:''' The table below indicates the chosen inoculation volume (two for each BioBrick) and the correct gel antibiotic concentration for all samples.(Extra caution was taken to allow agar to cool before adding Ampicillin, in case this is the cause of difficulty). 

{| class="wikitable"
|-
! colspan="2" |  Samples !! Volume Inoculated !! Antibiotic in Gel (ug/ml)
|-
| rowspan="4" |BioBrick ||rowspan="2" | BBa_C0040  || 10ul || rowspan="4" | Ampicillin(50ug/ml)
|-
| 90ul 
|-
| rowspan="2" | BBa_R0040  || 10ul 
|-
| 90ul 
|-
| rowspan="2" | Control || Positive (Contains BioBrick BBa_C0040)|| 36ul || No Antibiotic
|-
| Negative (No BioBrick)|| 36ul ||  1x Ampicillin(50ug/ml)
|-
|}


== Tuesday 31.8.12 ==

'''Aim - Check results of Transformation:''' The table below indicates whether there was growth on the Agar Plates after Transformation. 


{| class="wikitable"
|-
! colspan="2" |  Samples !! Volume Inoculated !! Colony Formation
|-
| rowspan="2" |BioBrick ||rowspan="2" | BBa_C0040  || 10ul || Yes
|-
| 90ul || Yes
|-
| rowspan="2" |BioBrick ||rowspan="2" | BBa_R0040  || 10ul || Yes
|-
| 90ul || Yes
|-
| rowspan="2" | Control || Positive (Contains BioBrick BBa_C0040)|| 36ul || Yes
|-
| Negative (No BioBrick)|| 36ul || No
|-
|}


'''Conclusion:''' Transformation was successful so will proceed to Colony Picking

'''Method'''

'''Aim - Picking colonies:''' 
 
<html><div class="protocol protocol-ColPic">Picking Colonies</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/ColPic}}<html></div></html>

'''Step 2 – Inoculating Colonies into a Selective Broth:''' The table below indicates the volume of broth and the concentration of antibiotic required for each BioBrick.

{| class="wikitable"
|-
! colspan="2" |  Samples !! Volume Inoculated !! Broth (ml) !! Antibiotic (ug/ml)
|-
| rowspan="8" |BioBrick ||rowspan="2" | BBa_C0040  || 10ul ||rowspan="8" |Lysogeny Broth (5) || rowspan="8" | Ampicillin(50ug/ml)
|-
| 90ul 
|-
| rowspan="2" | BBa_R0040 || 10ul 
|-
| 90ul 
|}


== Wednesday 1.8.12 ==


'''Aim – Results from Colony Picking'''

'''Results:''' The table below indicates whether there was growth for the BioBricks


{| class="wikitable"
|-
! colspan="2" |  Samples !! Volume Inoculated !! Growth
|-
| rowspan="8" |BioBrick ||rowspan="2" | BBa_C0040  || 10μl || Yes
|-
| 90μl || Yes
|-
| rowspan="2" | BBa_R0040  || 10μl || Yes
|-
| 90μl || Yes
|}


'''Conclusion:''' We can move on to miniprep

'''Methods'''

'''Miniprep of Samples'''

<html><div class="protocol protocol-Miniprep">Miniprep Protocol 1 (ANACHEM)</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Miniprep2}}<html></div></html>

'''Nanodrop'''

<html><div class="protocol protocol-Nanodrop">Nanodrop Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Nanodrop}}<html></div></html>

'''Results:'''
{| class="wikitable"
|-
! BioBrick !! λ260 !! λ 280
|-
| BBa_C0040 (ng/μl) || 9.4 || 18.9 
|-
| BBa_R0040 (ng/μl) ||9.3 || 36.9
|-
|}

'''Conclusion:''' The concentration of these plasmids is very low. We can conclude that the transformation has failed. The reasons for the continued failure of these plasmids are unclear

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== Tuesday 31.7.12 ==

'''Aim:''' to generate enough plasmid backbones  (pSB1K3, pSB1C3, pSB1A3) that we could use for 3A ligation. After the PCR reaction, we can detect the presence of the correct products by running a 1% gel electrophoresis.

'''Method:'''
<html><div class="protocol protocol-PCR">PCR Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/PCR}}<html></div></html>

== Wednesday 1.8.12 ==

'''Aim:''' to run a 1% gel of the products of yesterdays PCR reaction. 

'''Method'''

<html><div class="protocol protocol-Electrophoresis">Electrophoresis Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Electrophoresis}}<html></div></html>

'''Results:''' The image below shows a 1% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 8.2. Lane 1, 2 and 3 all show products of approximately 2000bp which is of the expected size for the plasmid backbones PSB1K3 (indicated by A), PSB1C3 (indicated by B) and PSB1A3 (indicated by C).


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{| class="wikitable"
|-
! Plasmid Backbone !! Expected Size bp
|-
| PSB1K3 (Kanamycin)  || 2000
|-
| PSB1C3 (Chloramphenicol)  || 2000
|-
| PSB1A3 (Ampicillin) || 2000
|}

'''Conclusion:'''The PCR was successful.'''



== Friday 3.8.12 ==

'''Aim:''' PCR clean up for the plasmid backbones and nanodrop. 

'''Methods:'''

PCR clean up

<html><div class="protocol protocol-Nanodrop">Nanodrop Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Nanodrop}}<html></div></html>


Results: Nanodrop concentrations are presented in the table below. All are low except for the backbone containing kanamycin. We concluded that PCR for these backbones needs to be repeated

{| class="wikitable"
|-
! BioBrick !! λ260 !! λ 280
|-
| PSB1A3 (ng/μl) || 27.3  || 26.6  
|-
| PSB1C3 (ng/μl) || 23 || 24.4 
|-
| PSB1K3 (ng/μl) ||66.5  || 87 
|-
|}


'''Conclusion:''' The PCR for PSB1A3 and PSB1C3 need to be repeated. 

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==Tuesday 31.7.12==

'''Aim:''' In order to set up our salt tolerance characterisation we must grow up 10ml of W3110 and Salt Tolerance (BBa_K398108) transformed W3110 overnight



== Wednesday 1.8.12 ==
'''Aim:''' Today we commence the protocols for characterising salt tolerance. 

'''Results:''' Our results demonstrate that out transformed cells are salt tolerant, compared to the untransformed W3110 cells.Our results reflect those of TU Delft 2010 who characterised the same BioBrick. However they did not show their results beyond the exponential phase, which we have included. 

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'''Conclusions:'''
Growth rate during exponential phase is greater in the K398108 transformed E.coli W3110 than in the untransformed (same conclusion as drawn by TU Delft 2010).  Increase in growth rates significantly lower than original at highest salt concentration – could be explained by use of different E.coli strains i.e. higher growth rate of the non-transformed strain. We plan to repeat to strengthen conclusions. 


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== Wednesday 1.8.12 ==


'''Aim:''' Start 3A assembly to ligate the first (BBa_J23119+BBa_B0034), and second (plasmid backbone PSB1K3 and Pcst starvation promoter) constructs. 


'''Method'''

==Thursday 2.8.12 ==

'''Aim:''' Evaluation of the plates from 3A ligation. This should inform us as to whether we can proceed and do the colony picking of both A (J23119+BB0034) and B (Pcst + BB0034) constructs.

 
'''Result:''' Observations showed that 3A was successful for the construct A, as shown in the image below. However there was no growth assembled pCST+BBa_B0034 (construct B). The following table summarises the results.

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'''Conclusion:''' Colony picking was carried out for the first construct (BBa_J23119+BBa_B0034), but not from the second construct (plasmid backbone PSB1K3 and Pcst starvation promoter). 

'''Method'''

<html><div class="protocol protocol-ColPic">Picking Colonies Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/ColPic}}<html></div></html>

== Friday 3.8.12 ==

'''Aim:'''Check the results of colony picking.

'''Results:''' There was growth. Next we can proceed to run a gel and a nanodrop to detect the presence of the correct bands, and whether we have a useable concentration. 




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== Thursday 2.8.12 ==

'''Aim - Transformation of alternative BioBricks:''' We have colony picked the BBa_J23119 constitutive promoter and the BBa_B0034 Ribosome Binding Site twice (Expt 6.3 & Expt 7,3), and gained a reasonable plasmid concentration. However the concentration was not high enough to be able to detect the product on the gel. This concerns us, because although we have detected the correct plasmid backbone, we are worried that we lack evidence that the transformation was successful. One option is to attempt several other BioBricks with the same function, in order to increase the chances of generating a product on the gel - and confirming that we definitely have a BioBricks with the required function.It would also increase the chance of generating successful ligations if we can reach a higher concentration of plasmid. We will also be reattempting the transformation of BBa_I750016 which failed several attempts at colony picking. 


'''Method:'''
<html><div class="protocol protocol-Transformation">Transformation Protocol 1</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Transformation1}}<html></div></html>


'''Step 1 – Thawing Cells:''' Use W3110 cell line created in Week 2 (Expt 2.1)

'''Step 3 – Addition of BioBrick:''' To each 2ml eppendorf, add 1ul of the following BioBricks. Include an extra tube as a control, with no BioBrick added

{| class="wikitable"
|-
!  colspan="2" |   !! Function !! Module
|-
| rowspan="4" |BioBrick|| BBa_J23100 || Constitutive Promoter || All
|-
| BBa_J23106 || Constitutive Promoter || All
|-
| BBa_B0030 || Ribosome Binding Site || All
|-
| BBa_I750016 || Ribosome Binding Site || All
|-
| rowspan="2"|Control || Positive (one for each of the above BioBricks)|| ||
|-
| Negative (No BioBrick) || ||
|}

'''Step 9 – Plating samples on Agar Plates:''' The table below indicates the chosen inoculation volume (two for each BioBrick) and the correct gel antibiotic concentration for all samples.(Extra caution was taken to allow agar to cool before adding Ampicillin, in case this is the cause of difficulty). 

{| class="wikitable"
|-
! colspan="2" |  Samples !! Volume Inoculated !! Antibiotic in Gel (ug/ml)
|-
| rowspan="8" |BioBrick ||rowspan="2" | BBa_J23100  || 10ul || rowspan="8" | Ampicillin(50ug/ml)
|-
| 90ul 
|-
| rowspan="2" | BBa_J23106  || 10ul 
|-
| 90ul 
|-
| rowspan="2" | BBa_B0030 || 10ul 
|-
| 90ul 
|-
| rowspan="2" | BBa_I750016 || 10ul 
|-
| 90ul 
|-
| rowspan="2" | Control || Positive || 36ul || No Antibiotic
|-
| Negative (No BioBrick)|| 36ul ||  1x Ampicillin(50ug/ml)
|-
|}

== Friday 3.8.12 ==

'''Aim - Check results of Transformation:''' The table below indicates whether there was growth on the Agar Plates after Transformation. Included below are images of the Agar Plates for each BioBrick.


{| class="wikitable"
|-
! colspan="2" |  Samples !! Volume Inoculated !! Colony Formation
|-
| rowspan="8" |BioBrick ||rowspan="2" | BBa_J23100  || 10ul || Yes
|-
| 90ul || Yes
|-
| rowspan="2" | BBa_J23106  || 10ul || Yes
|-
| 90ul || Yes
|-
| rowspan="2" | BBa_B0030  || 10ul || Yes
|-
| 90ul || Yes
|-
| rowspan="2" | BBa_I750016  || 10ul || Yes
|-
| 90ul || Yes
|-
| rowspan="2" | Control || Positive (Contains BioBrick BBa_C0040)|| 36ul || Yes
|-
| Negative (No BioBrick)|| 36ul || Yes
|-
|}


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'''Conclusion:''' We had growth for all of our BioBricks, but also for the negative control which indicates there was contamination. Given we have had several issues with negative control recently, we decided we would not let the contamination delay the project. We will risk colony picking with the hope that we have some correctly transformed colonies. This will be commenced next week. It is worth noting that for Constitutive Promoter BBa_J23106 and for the Gas Vesicle Cluster BBa_I750016 there was a continuous film of growth. This suggests to us that the agar was not selective enough.



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