User:Chris D Hirst/Plasma

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m (Restriction Enzyme, ORF and Primer Analysis)
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Before starting, you decide to ''in silico'' test your cloning stratergy.
Before starting, you decide to ''in silico'' test your cloning stratergy.
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====Resources====
====Resources====
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Import a vector containing BBa E0240 from this [http://www.openwetware.org/wiki/Image:BBa_E0240.pof .pof file]
Import a vector containing BBa E0240 from this [http://www.openwetware.org/wiki/Image:BBa_E0240.pof .pof file]
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===Restriction Enzyme, ORF and Primer Analysis===
===Restriction Enzyme, ORF and Primer Analysis===
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Add the primers from [http://www.openwetware.org/wiki/Media:Plasma_Tutorial_Primers.txt this text file] to this project on PlasmaDNA (using the add primer button not enter sequence button), then look at the two outputs under primer analysis.  
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Add the primers from [http://www.openwetware.org/wiki/Media:Plasma_Tutorial_Primers.txt this text file] to this project on PlasmaDNA (using the add primer button not enter sequence button - '''Make sure you enter the binding sequence and 5' sequence correctly!'''), then look at the two outputs under primer analysis.  
*Can you suggest a use for SeqF and SeqR?
*Can you suggest a use for SeqF and SeqR?
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===Restriction Cloning - Biobricks RFC 10 (BBa K116617)===
===Restriction Cloning - Biobricks RFC 10 (BBa K116617)===
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The first step is clonoy PCR - simply put, PCR is carried out on all the DNA from a colony (obtained by boiling) to look for a correctly sized Multiple Cloning Site (MCS) - ie. the prefix, suffix and everything inbetween! Carry out PCR on the ligated fragment using the pSBF and pSBR primers using the PCR window. Is the fragment approximately the right length for K116617?
The first step is clonoy PCR - simply put, PCR is carried out on all the DNA from a colony (obtained by boiling) to look for a correctly sized Multiple Cloning Site (MCS) - ie. the prefix, suffix and everything inbetween! Carry out PCR on the ligated fragment using the pSBF and pSBR primers using the PCR window. Is the fragment approximately the right length for K116617?
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*Is there any 'cheat' teachnique you could use to help you select for a correctly assembled DNA?
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For easy selection of correct transformants you decide it would be better to transform into a vector with a resistance other than Amp or Kan so choose to use [http://partsregistry.org/Assembly:3A_Assembly 3A Assembly].
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To construct the I13018 past of the F2620 testing construct (simialr to T9002), it is necessary to combine BBa R0040 and BBa I0462.
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For easy, speedy selection of correct transformants you decide it would be better to transform into a vector with a resistance other than Ampicillin when making the first half of the construct (I13018). By transforming by [http://partsregistry.org/Assembly:3A_Assembly 3A Assembly] into pSB1C3, correct transformants will be resitant to Chlorampheniol rathern than Ampicillin.
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To assemble this, R0040 and I0462 must be cut ready for insertion into the plasmid back bone. In particular, R0040 mustbe cut front insert and I0462 must be cut back insert. The sequences of the two BioBrick can be found in this [http://www.openwetware.org/wiki/Image:I13018_Compomnents.pof .pof file].
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A vector is required to clone the pair of inserts. Plasmid backbones are now supplied along with the registry distribution and can be amplified by PCR. For submission, pSB1C3 is preferred so this should be amplified using the pSB-3P and pSB-2Ea primers. This amplified backbone can now be cut with EcoRI and PstI to allow ligation of the three pieces.
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*What extra step between the PCR amplification and the EcoRI and PstI digestion would be required if this was carried out in lab?
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===Cloning - In-Fusion (Similar to T9002)===
===Cloning - In-Fusion (Similar to T9002)===

Revision as of 12:24, 13 July 2010

Contents

Introduction


PlasmaDNA[1] is a free to use computer programme developed by the University of Helsinki to allow the easy mainpulation of DNA in silico. In the hands of an experience user, it can be used to fully plan out the assembly of a synthetic construct from base parts, genome sequences, cDNAs - basically any DNA input for which a text or FASTA file can be found. The ability to attempt a cloning strategm in silico before going into the lab can help identify bottlenecks and critical paths while allowing the user to visually see what they are producing.


While other programmes can do this, PlasmaDNA is free, very use friendly and produces highly detailed plasmid maps in 3 flavours. Some lab members already create PlasmaDNA (.pof) files for all the vectors and constructs they build. This and the ability to visualise cloning can prove very useful to aid understanding and debugging when issues arise.

PlasmaDNA can be accessed here.

Tutorial


If you follow this tutorial, you should be able to use most of the features of PlasmaDNA. If you get stuck at any point, try having a play around with the programme! Most difficulties can easily be solved by trial and error. If you don't understand what you are doing, let me know and I'll help you.


Be aware that though the story behind this is hypothetical (ie. fictional) it is not implasuible. The registry has improved it's Quality Control so should now be far more reliable!


Your lab needs to produce and re-characterise BBa F2620 under a new set of conditions but has lost the original DNA of the testing construct (similar to BBa T9002). To make matters worse, someone has been prepping samples without keeping a back-up stock with the distribution, meaning that some of the ideal building components are missing. Fortunately there are other similar intermediates yet to be prepped that you can use to rebuild this.


Your supervisor reminds you that you are short on time so you decide to use three different methods (Standard BioBricks Assembly, 3A Assembly and Clonetech™ In-Fusion[2] - Figure 1 in this manual gives a good overview) to speed up you workflow.


Another student has attempted to get BBa F2620 from the registry plate and succesfully transformed the cells but upon gel analysis all their transformants contain an empty plasmid! It will be quicker therefore to make your test construct in two halves - BBa I13018 and BBa K116617 - and assemble them into the expression vector later.


Before starting, you decide to in silico test your cloning stratergy.


Resources

You have access to all the consumables you will need and the following BioBricks:

BBa R0040

BBa I0462

BBa R0062

BBa E0240

BBa pSB1C3 Backbone

Standard Expression Vector


Walkthrough

PlasmaDNA Control Panel
PlasmaDNA Control Panel

Importing Sequences


PlasmaDNA can import sequences in one of three formats: Plain text, FASTA (similar to plain text) and .pof (Plasma) files.


To start off this tutuorial, enter the sequence of BBa R0062 (from the parts registry) and remember to add the BioBrick prefix and suffix!


Import a vector containing BBa E0240 from this .pof file


Restriction Enzyme, ORF and Primer Analysis


The Output viewer should already be open. If not, open it (button in the top left of the control panel) and look at the output of the two inputs. One should be a complete circular vector and one should be a short linear piece. The view is currently set to restriction analysis - ie. it shows all the restriction enzyme cutting sites on the sequences. Two alternative viewer modes are ORF analysis - this looks for Open Reading Frames (ie. coding regions) in the sequence according to a user set threshold (default is generally ok) and primer analysis - compares the input sequence to known primer sequences.


There are currently two known ORFs in E0240 and none in R0062, however the ORF analysis function indicates there is an extra coding region here. Determine the nature of the coding region - either by part knowledge or using the blast function in the ORFs menu and add it to the current project.


Add the primers from this text file to this project on PlasmaDNA (using the add primer button not enter sequence button - Make sure you enter the binding sequence and 5' sequence correctly!), then look at the two outputs under primer analysis.


  • Can you suggest a use for SeqF and SeqR?


Restriction Cloning - Biobricks RFC 10 (BBa K116617)


The first step, addition on R0062 and E0240 to make K116617 should be achieved using Standard BioBrick Assembly as this will prevent scars and can be done in parallel to production of the other half.


Restriction Cloning should be carried out based on a procedure of Digestion to generate relevant insert and vector fragments and ligation together (in lab, gel purification and PCR purification - to remove enzymes - would also be carried out between the digestion and the ligation).


  • What enzymes will R0062 and E0240 need to be cut with for them to go together correctly?


Digest R0062 and E0240 (Digest! button) with the appropriate enzymes and select (effectively in silico purify) the correct fragment to keep. Now open the ligation window and attempt to ligate the two parts together.


  • What is the final lab step in cloning before selection of correctly assembled products which isn't needed in silico?


Analysis of cloning results


The output of the ligation will be a plate of transformed cells. It is not necessarily true that all the transformants will contain correctly assembled DNA (in many cases, the majority will be incorrect - often 'background' ie. undigested or re-ligated vector. Usually a 'background plate' will also be transformed to give an idea of the numbers). It is therefore necessary to select for the correctly assembled DNA.


With a large number of transformants, a three step approach is the most appropriate (with a small number, skipping step one and going straight to mini-prep and gel analysis may be mroe appropriate).


The first step is clonoy PCR - simply put, PCR is carried out on all the DNA from a colony (obtained by boiling) to look for a correctly sized Multiple Cloning Site (MCS) - ie. the prefix, suffix and everything inbetween! Carry out PCR on the ligated fragment using the pSBF and pSBR primers using the PCR window. Is the fragment approximately the right length for K116617?


  • Is there any 'cheat' teachnique you could use to help you select for a correctly assembled DNA?


The second step is to mini-prep (lab only - grow up cells and extract plasmid DNA) and then use a restriction digest to check that the insert and vector are the correct sizes.


  • How many enzymes would it be useful to use in this step and which ones would you choose?


Digest the ligated fragment using the digest button as before. Do the sizes of the vector and insert match what they should be?


The final step is the most important. Mutations occaisonally occur and can alter the function of your part or device if you are unlucky, therefore all parts that are made should be sequenced to ensure there has been no mutations (particularly non-silent mutations). Open the fragment and sequence information window and perform an Align BLAST Search[3] against the sequence of BBa K116617. Have any errors been picked up? If so are they important?


It may be useful for you to delete old, no longer needed fragments, or to re-name new fragments to stop confusion later. Both these functions can be carried out in the fragment sequence and information menu (Orange box in above diagram). Once you are sure you have validated that the part is correct, delete the old fragments and label the new ligation K116617.


3A Assembly - PCR and Biobricks (BBa I13018 )


To construct the I13018 past of the F2620 testing construct (simialr to T9002), it is necessary to combine BBa R0040 and BBa I0462.


For easy, speedy selection of correct transformants you decide it would be better to transform into a vector with a resistance other than Ampicillin when making the first half of the construct (I13018). By transforming by 3A Assembly into pSB1C3, correct transformants will be resitant to Chlorampheniol rathern than Ampicillin.


To assemble this, R0040 and I0462 must be cut ready for insertion into the plasmid back bone. In particular, R0040 mustbe cut front insert and I0462 must be cut back insert. The sequences of the two BioBrick can be found in this .pof file.


A vector is required to clone the pair of inserts. Plasmid backbones are now supplied along with the registry distribution and can be amplified by PCR. For submission, pSB1C3 is preferred so this should be amplified using the pSB-3P and pSB-2Ea primers. This amplified backbone can now be cut with EcoRI and PstI to allow ligation of the three pieces.


  • What extra step between the PCR amplification and the EcoRI and PstI digestion would be required if this was carried out in lab?


Cloning - In-Fusion (Similar to T9002)



Exporting



References


  1. Angers-Loustau A, Rainy J, and Wartiovaara K. . pmid:17868482. PubMed HubMed [1]
  2. CloneTech™ In-Fusion [2]
  3. Altschul SF, Gish W, Miller W, Myers EW, and Lipman DJ. . pmid:2231712. PubMed HubMed [3]
All Medline abstracts: PubMed HubMed
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