User:Chris D Hirst/Plasma

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 debug 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.

Resources

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

BBa R0040

BBa I0462

BBa R0062

BBa E0240

BBa F2620

BBa pSB1C3 Backbone

Standard Expression Vector

Walkthrough

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, load 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 vactor 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), 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)

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 expressino vector later.

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

3A Assembly - PCR and Biobricks (BBa I13018 )

Oh no! You've attempted to get BBa F2620 from the registry plate and succesfully transformed the cells but upon gel analysis it appears that all your transformants contain an empty plasmid!

Luckily you find that BBa I0462 and BBa R0040 are correct upon prepping from the registry, though R0011 for some reason is in plasmid pSB1AK3 not pSB1A2.

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 3A Assembly.

References

1. Angers-Loustau A, Rainy J, and Wartiovaara K. PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories. BMC Mol Biol. 2007 Sep 17;8:77. DOI:10.1186/1471-2199-8-77 |

2. CloneTech™ In-Fusion