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Team:ZJU-China/Tools
From 2014.igem.org
GS-Box
A simple & efficient tool for Gene-Socket
Every excellent technique should be simple and efficient to use! As an excellent technique, gene-socket also does well in this respect------ it has its' own optimization tool: GS-BOX. GS-BOX is a bioinformatics tool which is designed for customs to build their own gen-sockets easily and efficiently. What need the customs do with the tool? Just input the gene circuit sequences and then GS-BOX will design the experimental process automatically and every detail that customs will need to use!
Service
GS-Box provides 3 kinds of modes and 7 services.
For mode 1, GS-Box can provide:
i. Homeoregion's sequence
ii. P-primer's sequence
iii. P-primer's GC percent
iv. P-primer's TM value
v. F-primer's sequence
vi. F-primer GC percent
vii. F-primer TM value
For other modes, it cotains two homeoregions and also two P-primers and F-primers.
If you dont know how to use these sources, please read the example below or read our projectcarefully.
Design Principle
Homeoregions:
1. Don't have similar sequences with E.coli
(to avoid off-target effects)
2. Don't have similar sequences with custom's parts
(to avoid off-target effects)
3. Fit the subsequent steps' requiring
Primer:
1. GC content & TM value
(to ensure that PCR can work. If the GC percent is between 40% -60%, the PCR result will be fine)
2. Duplex formation and hairpin
(to ensure that PCR can work. If the primer has duplex formation and hairpin, the PCR cannot get result)
3. Proper length
(to ensure that PCR can work. If the length is too short, the PCR cannot get the result; If the length is too long, the TM value will be very high and make PCR's result not good)
Design Method
Step 1. Generate more than 1000 sequences which contains 40 bp and are made of A/T/G/C
Step 2. Do blast between the sequences and the E.coli's genome to delete some sequences which have samilar sequences with E.coli. As a result, a database which has 1000 sequences was established.
Step 3. Do blast between the custom's sequences and the database's data to delete some sequences which have samilar sequences with custom's sequences. As a result, a secondary database was established.
Step 4. Select some data in the secondary database which has proper primer.
Step 5. Print out the primer's information.
How to use
GS-Box has 3 modes.
If you want to build a circuit which doesn't contian a promoter or terminator inside the circuit, you shuld choose mode 1;
If you don't want to use our promoter, you shuld choose mode 2;
If your circuit contians a terminator, you shuld choose mode 3;
For more information, please see the solutions below.
Help
You should know that if you choose a mode, this mode will be used for all of your parts. So if you just want to use the special mode for only one of your parts, you should use GS-Box twice, once without the special part and once only contain the special part.
For more information, please see the example below.
If you still have any questions when using GS-Box, please contact us.
Team Forum(BBS):
ZJU-China 2014 Team Forum on ZJU Sever
Post Address:
Biolab Center Room 413, ZJU Zijin'gang Campus, Yuhang Tang Road No.866, Hangzhou, Zhejiang
Email Address:
zjuchina2014 @ 163.com
Solutions
As you see, we built 2 parts to help biologists to insert their gene of interest( GOI ) into the chromosome. When we built this tool, we thought about a question: can Gene-Socket only insert simple genes like GFP into chromosome? Now, we can announce that the answer is "No". With the help of Gene-Socket, we can build kinds of gene circuits no matter how complex they are.
If we just want to insert one part into the chromosome, what should we do? We can add two homeoregions next to the part, then link them to the part BBa_k1433009. In this way, we can use Gene-Socket to insert the new gene-combination into chromosome. (See how we solve the problem in the picture). We call this occasion "Mode 1".
Now we want to build a more complex circuit. We want to insert a promoter, an RBS and a CDS. If we still use the old method, our promoter will not work. It seems that we need to find a new way. What if we change the homeoregions? We can replace the old promoter with a new one by designing the homeoregions. (See how we solve the problem in the picture). We call this occasion "Mode 2".
Well, what if our circuit contains a terminator? It will stop the gene expression and break our hearts. But if we link the circuit with a new part, BBa_k1433010, the problem seems to be solved. (See how we solve the problem in the picture). We call this occasion "Mode 3".
As you see, If you want to use GS-Box to insert your part into the chromosome,there are 3 classical mode for you.
- Mode 1: You choose to use the our official promoter & terminator
- Mode 2: Your don't want to use our promoter
- Mode 3: Your part contains a terminator
With the methods that we have used above, we can solve most problems when we want to insert a gene-circuit into chromosome, but sometimes the situation may be more complex. We cannot list all of the situations for you, but we have given you the tool and the idea to solve a new problem. Join the designers’ team and enjoy it!
Example
Example 1:
I just want to insert a simple gene---GFP(see the picture below). This situation is very simple. According to "Solutions", this is the mode 1, and I need to modify it as the picture below.
Example 2:
I want to insert a circuit, which contains a promoter, a RBS and GFP(see the picture below). According to "Solutions", this is the mode 2, and I need to modify it as the picture below.
Example 3:
I want to insert a circuit which contains a RBS, a GFP and a terminator(see the picture below). According to "Solutions", this is the mode 3, and I need to modify it as the picture below.
Download
GS-Box is an useful web tool, it contains some fuctions that people often use. So we share all the codes of GS-Box(except images) to help other IGEMers to use GS-Box in their own computers or share with more people. As you see, we submit a ZIP named "all codes" below and you can download it for free. In the ZIP, there is a "Read me" txt, please read it before you use it. You can also download our database which is strictly chosen and the data is OK to use. Before you use the codes and the database, you need to install a local-BLAST, which is developed by NCBI.
